WO1998008540A1 - Multivalent vaccine for clostridium botulinum neurotoxin - Google Patents

Multivalent vaccine for clostridium botulinum neurotoxin Download PDF

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Publication number
WO1998008540A1
WO1998008540A1 PCT/US1997/015394 US9715394W WO9808540A1 WO 1998008540 A1 WO1998008540 A1 WO 1998008540A1 US 9715394 W US9715394 W US 9715394W WO 9808540 A1 WO9808540 A1 WO 9808540A1
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WIPO (PCT)
Prior art keywords
toxin
protein
antibodies
difficile
recombinant
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PCT/US1997/015394
Other languages
French (fr)
Inventor
James A. Williams
Bruce S. Thalley
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Ophidian Pharmaceuticals, Inc.
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Publication date
Priority claimed from US08/704,159 external-priority patent/US6967088B1/en
Application filed by Ophidian Pharmaceuticals, Inc. filed Critical Ophidian Pharmaceuticals, Inc.
Priority to EP97940746A priority Critical patent/EP1105153A4/en
Priority to CA002296765A priority patent/CA2296765A1/en
Priority to AU42450/97A priority patent/AU4245097A/en
Publication of WO1998008540A1 publication Critical patent/WO1998008540A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the isolation ol polypeptides de ⁇ ved from Clostridium boiulimtm neurotoxins and the use thereof as immunogens for the production of vaccines, including multivended vaccines, and antitoxins.
  • Clostridium The genus Clostridium is comprised of gram-positive, anaerobic, spore-torming bacilli
  • the natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous: they are commonly found in soil. dust, sewage, marine sediments. c ,t, r P.H A Sneath et al . "Clostridium.” Bcrgey s Manual * of Systematic . Vol 2. pp 1 141 - 1200. Williams & ⁇ ilktns ( 1986).) Despite the identification ot approximate! 100 species ol Closiridiu .
  • Botulism disease mav be giouped into lout tvpes based on the method ot introduction ot toxin into the bloodstream 1 ood-bome botulism results Irom ingesting ⁇ nproperlv pteseivcd and madequatelv heated food that contains botuhnal toxm there were " > -> cases ot lood-botne botulism in the United States between 1976 and 1984
  • Inhalation botulism results when the tox is inhaled Inhalation botulism has been icported as the lesult ol accidental exposuie in the laboratorv
  • the intestinal environment is not as acidic, allow ing for ( ' . hoiulinum spore germination, growth, and toxin production.
  • some adults who have undergone antibiotic therapy which alters intestinal microtlora become more susceptible to botulism.
  • Type C toxin affects waterfow l, cattle, horses and mink.
  • Type D toxin affects cattle, and type E toxin affects both humans and birds.
  • ⁇ trivalent antitoxin derived from horse plasma is commercially available from
  • a pentavalent human antitoxin has been collected from immunized human subjects for use as a treatment for infant botulism.
  • the supply of this antitoxin is limited and cannot be expected to meet the needs of all indiv iduals stricken w ith botulism disease.
  • I n addition, collection ot human sera must involve screening out HIV and other potentially serious human pathogens. ( P.J. Schwarz and S.S. Arnon. Western .1. Med. 1 56: 197 ( 1 992 ).
  • SI DS Sudden I nfant Death Syndrome
  • crib death SI DS. also known as crib death.
  • SI DS is officially recognized as infant death that is sudden and unexpected and that remained unexplained despite complete post-mortem examination.
  • the link of SIDS to infant botulism came when fecal or blood specimens taken at autopsy from SI DS infants were found to contain C hoiulinum organisms and/or toxin in 3-4% of cases analyzed. [D.R. Peterson et al . Rev. Infect. Dis. 1 :630 ( 1979). ]
  • 160 healthy infants 0.6%) had C. hoiulinum organisms in the leees and no botulinal toxin.
  • S. Arnon et al.. Lancet, pp. 1 273-76. June 1 7. 1978. S. Arnon et al.. Lancet, pp. 1 273-76. June 1 7. 1978.
  • SI DS is the number one cause of death in children between one month and one year old. ( S. Arnon et al.. Lancet, pp. 1 273-77. June 1 7. 1978.) More children die from SIDS in the first vear than from anv other simile cause of death in the First fourteen years of life. In the United States, there are 8.000-10.000 SIDS victims annually. Id
  • C difficile is a part of the normal gastrointestinal flora of 2-10% ot humans. [G F Brooks el al .
  • C. difficile is commonly associated w ith nosocomial infections.
  • the organism is often present in the hospital and nursing home environments and may be carried on the hands and clothing of hospital personnel who care for debilitated and immunocompromised patients.
  • As many of these patients are being treated w ith antimicrobials or other chemotherapeutic agents, such transmission of ( ' . difficile represents a significant risk factor for disease. ( Engelkirk et al.. pp. 64-67.) C.
  • C. difficile is associated w ith antimicrobial therapy with virtually all antimicrobial agents (although most commonly ampiciilin. clindamycin and cephalosporins). It is also associated w ith disease in patients undergoing chemotherapy with such compounds as methotrexate. 5- 20 lluorouracil. cyclophosphamide. and doxorubicin. [ S.M. Finegold el al.. Clinical iiuide lo
  • I igure 2 shows the IgY antibody titer to ( ' hoiulinum type A toxoid in eggs, measured bv 1 I ISA
  • I igure 3 shows the results of C difficile toxm A neutralization assav s
  • Figure 4 shows the results oi ( ' difficile toxin B neutralization assav s I igure 5 shows the results o ⁇ C difficile toxin B neutralization assays
  • I iguie 6 is a lest ⁇ ction map ot C difficile tox A gene, showing sequences ol primers 1-4 (SEQ ID NOS- 1-4)
  • I igure 7 is a Western blot ol ( ' difficile tox A leaclive protein
  • I igure 8 shows ( difficile toxm A expression constructs.
  • I igure 9 shows C difficile toxin A expression constructs
  • 1 igure 10 shows the purification ot recombinant ( ' difficile tox A
  • I iguie 12 shows the results for a ( ' difficile toxm A neutralization plate
  • I iguie 13 shows the results for a C difficile toxin A neutralization plate
  • I iguie 15 shows ( difficile toxin A expression constructs
  • I iguie 16 shows a chromatograph plotting absorbance at 280 nm against letention time loi a pMAl 870-680 IgY PI (i preparation.
  • I igure 17 shows two recombinant ( ' difficile toxm B expression constructs
  • figuie 18 shows ( ' difficile toxin B expression constructs
  • Figure 19 shows ( ' difficile toxm B expression constructs
  • I igure 20 shows ( ' difficile toxin B expression constructs
  • I igure 23 shows ( difficile toxin B expression constructs
  • 1 igure 24 is a Western blot ol ( difficile toxin B reactive piotem Figure 25 shows ( hoiulinum t pe A toxin expression constructs constructs used to piovidc ( hoiulinum or ( difficile sequences are also shown
  • Figuie 26 is an SDS-PAGF gel stained with Coomaisse blue showing the purification ol iccombinant ( botulinum tvpe A toxin fusion proteins
  • Figuie 27 shows ( botulinum tv pc A toxm expression constructs constiucts used to piovidc ( hoiulinum sequences are also shown
  • I iguie 28 is an SDS-PAGT gel stained with ( oomaisse blue showing the purification of pIlisBot piotetn using the Ni-NI ⁇ lesin
  • I iguie 29 is an SF)S-P ⁇ GE gel stained with Coomaisse blue showing the expression of pIlisBot protein in BI 21(1X3) and BF21(DF 3)pl vsS host cells
  • I igme 30 is an SDS-PAGF gel stained with Coomaisse blue showing the purification ol pliisBol piotem using a batch absorption procedure
  • 1 iguie 31 is an SDS-P ⁇ GE gel stained with Coomaisse blue show inn the potcation ol pliisBol and pll ⁇ sBot(nat ⁇ ve) pioteins using a Ni-N I ⁇ column
  • I igure 32 is an SDS-PAGI gel stained with Coomaisse blue show mi: the purification ol pllisBol ⁇ protein expressed in pll ⁇ sBotA(svn) kan laclq 17/p C YC Gro/BI 2KDE3) cells using an IDA column
  • I iguie 33 is an SDS-PAGF gel stained with C omaisse blue showing the purification ol pHisBot ⁇ pHisBotB and pllisBotE proteins bv ID ⁇ chromatographv followed bv chiomatogiaphv on S-100 to lemove tolding chaperones
  • I igure 34 is an SDS-PAGE gel stained with Coomaisse blue showing the extracts derived tiom pHisBotB amp T7lac/BL21(Dr3) cells betoie and after purification on a Ni- N ⁇ A column
  • Figure 35 is an SDS-PAG1 gel mn under native conditions and stained with C oomaisse blue showing the removal ol folding chaperones tiom IDA-purified BotB protein usirm a S-1 0 column
  • Figure 36 is an SDS-PAGE gel stained with Coomaisse blue showing proteins that eluted during an imidazole step gradient applied to a IDA column containing a lysate of pHisBotB kan laclq T7/pACYCGro/BL21 (DE3) cells.
  • Figure 37 is an SDS-PAGE gel run under native conditions and stained w ith Coomaisse blue showing IDA-purified BotB protein before and after ultrafiltration.
  • Figure 38 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of BotE protein using a NiNTA column.
  • F igure .39 is an SDS-PAGE gel stained with Coomaisse blue showing extracts derived from pHisBotA kan 17 lac/BL21 (DE3) pLysS cells grown in fermentation culture.
  • F igure 40 is a chromatogram show ing proteins present after I DA-purified BotE protein was applied to a S- 100 column.
  • neutralizing is used in reference to antitoxins, particularly antitoxins comprising antibodies, w hich have the ability to prevent the pathological actions of the toxin against which the antitoxin is directed.
  • the term "overproducing" is used in reference to the production of clostridial toxin polypeptides in a host cell and indicates thai the host cell is producing more of the clostridial toxin by virtue of the introduction of nucleic acid sequences encoding said clostridial toxin polypeptide than would be expressed by said host cell absent the introduction of said nucleic acid sequences.
  • the host cell express or overproduce said toxin polypeptide at a level greater than 1 mg/liter o ⁇ host cell culture.
  • a host cell capable of expressing a recombinant protein at a level greater than or equal to 5% of the total cellular protein is a host cell in which the recombinant protein represents at least 5% of the total cellular protein. To determine what percentage of total cellular protein the recombinant protein represents, the following steps are taken. A total of 10 C)D Mschreib, units of recombinant host cel ls (e tc..
  • 1 7 - pellets are resuspended in 1 ml of 50 mM NaHPO.,, 0.5 M NaCl. 40 mM imidazoie buffer ( pH 6.8) containing 1 mg/ml lysozyine. The samples are incubated for 20 min at room temperature and stored ON at -70°C. Samples are thawed completely at room temperature and sonicated 2 X 1 0 seconds w ith a Branson Sonifier 450 microtip probe at # 3 power setting. The samples are centrifuged for 5 min. at maximum rpm in a microfuge. An aliquot
  • Coomassie blue it is not necessary that Coomassie blue be employed for the detection of protein, a number of fluorescent dyes ⁇ e. ⁇ .. Sypro orange S-665 1 ( Molecular Probes. Eugene. OR] may be employed and the stained gel scanned using a lluoroimager
  • a host cell capable of expressing a recombinant protein as a soluble protein at a level greater than or equal to 0.25% of the total soluble cellular protein is a host cell in which the amount of soluble recombinant protein present represents at least 0.25% of the total cellular protein.
  • total soluble cellular protein refers to a clarified PEI lysate prepared as described in Example l (c )( iv). Briefly, cells are harvested follow ing induction of expression o ⁇ recombinant protein ( at a point of maximal expression ). The cells are resuspended in cell resuspension buffer (CRB: 50 mM NaPO,. 0.5 M NaCl.
  • PEI clarified lysate The recombinant protein present in the PEI clarified ly sate is then purified ⁇ e.g., by chromatography on an IDA column for his-tagged proteins). The amount of purified recombinant protein (i. e.. the eluted protein) is divided by the concentration of protein present in the PL- I clarified lysate (typically 8 mg/ml when using a 20% cell suspension as the starting material ) and multiplied by 1 00 to determine what percentage of total soluble cellular protein is comprised of the soluble recombinant protein ( see Example
  • fusion protein refers to a chimeric protein containing the protein of interest (i. e.. C. hoiulinum toxin A. B. C. D. E. ! ⁇ ' . or G and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-toxin protein).
  • the fusion partner may enhance solubility of the C. hoiulinum protein as expressed in a host cell, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both.
  • the fusion protein may be remov ed from the protein of interest (i. e...
  • non-toxin protein or “non-toxin protein sequence” refers to that portion of a fusion protein which comprises a protein or protein sequence w hich is not deriv ed from a bacterial toxin protein.
  • protein of interest refers to the protein whose expression is desired w ithin the fusion protein, in a fusion protein the protein of interest w ill be joined or fused with another protein or protein domain, the fusion partner, to allow for enhanced stabilit of the protein of interest and/or ease of purification of the fusion protein.
  • maltose binding protein refers to the maltose binding protein o ⁇ / • . ' . coli.
  • a portion of the maltose binding protein may be added to a protein of interest lo generate a fusion protein: a portion of the maltose binding protein may merely enhance the solubility of the resulting fusion protein when expressed in a bacterial host.
  • a portion of the maltose binding protein may allo affinity purification of the fusion protein on an amylose resin.
  • poly-histidine tract when used in reference to a fusion protein refers to the presence of two to ten histidine residues at either the amino- or carboxy- terminus of a protein of interest. A poly-histidine tract o ⁇ six lo ten residues is preferred.
  • the poly -histidine tract is also defined functionally as being a number of consecutive histidine residues added to the protein of interest which allows the affinity purification of the resulting fusion protein on a nickel-chelate or IDA column
  • the removal ot non-immunoglobuhn proteins and/oi the removal oi immunoglobuhns that do not bind toxin results m an increase in the percent ot toxm-ieacti e immunoglobuhns in the sample
  • recombinant toxm polvpeptides are expressed in bacterial host cells and the toxm polvpeptides are purified bv the removal oi host ceil pioteins the percent ot recombinant toxin polypeptides is thereby incieased in the sample ⁇ dd ⁇ t ⁇ onall
  • I he te ⁇ n lecombmant DNA molecule as used herein reteis to a D ⁇ molecule which is comprised ol segments of DNA joined together bv means oi molecular biological techniques
  • 'native protein' refers to a protein which is isolated from a natuial souice as opposed to the pioduction of a protein bv iecombinant means ⁇ s used herein the teim portion' when in refeience to a piotem (as in 'a portion ol a given piotem") teteis to liagments ol that protein
  • the tiagments mav range m size Irom loin am o acid tesidues to the entire ammo acid sequence minus one ammo acid
  • ⁇ s used herein 'soluble when in reference to a protein pioduced bv iecombinant DNA technology a host cell is a piotem which exists in solution in the cvtoplasm of the host cell, it the piotem contains a signal sequence the soluble protein is exported to the penplasmic space in bactc ⁇ a hosts and is secreted into the cultuie medium in eucaryotic cells capable ol secretion oi bv bacterial host possessing the appropriate genes (/ e . the k gene) In contiast.
  • an insoluble piotem is one which exists in denatured foim inside cv toplas ic granules (called inclusion bodies) in the host cell
  • inclusion bodies High level expression (/ c greater than 10- 20 mg iecombinant piotem/htei ot baetenal culture)
  • ot tecombinani pioteins often lesults m the expiessed protein being lound in inclusion bodies in the baetenal host cells
  • ⁇ soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.
  • a soluble protein i e . a protein which w hen expressed in a host cell is produced in a soluble form
  • a "solubihzed" protein An insoluble i ecombinant protein found inside an inclusion body may be solubihzed (/ e . rendered into a soluble form ) by treating purified inclusion bodies with denaturants such as guamdine hy drochlonde.
  • SDS-solubih/ed inclusion body protein is soluble but not refolded
  • proteins which are soluble i e . dissolv ed
  • a solution dev oid ol significant amounts ot ionic detergents ( c tc . SDS ) or denaturants (c ,g . ui ea. guunidme hy drochloride)
  • proteins which exist as a suspension ol insoluble protein molecules dispersed within the solution A soluble protein will not be removed Irom a solution containing the protein by centrifugation using conditions suf ficient to remov e bacteria present in a liquid medium ( i e .
  • piotem is said to be soluble in the solution tested I I the majonty ot piotem B is found in the pellet (/ e . greater than 90%). then protein B is said to exist as a suspension in the solution tested
  • ect Ihe teim 'pvrogen as used herein refers to a fc ei -producing substance Pvrogens mav be endogenous to the host (c g prostaglandins) oi mav be exogenous compounds (e tc bacteria] endo- and exotoxins nonbacte ⁇ al compounds such as antigens and certain steroid compounds etc ) Ihe presence ol pvrogen in a pharmaceutical solution mav be detected using the I S Pharmacopeia (USP) rabbit fever test (United States Pharmacopeia. Vol XXII ( 1990) United States Pharmacopeial C onvention Rockville MD p 151)
  • I he teim endotoxin refers to the high moleculai weight complexes associated with the outer membrane ot gram-negati e bactciia
  • Unpunfied endotoxin contains lipids pioteins and carbohvdiates Highlv purified endotoxin does not contain piotem and is icleiied to as hpopolv saccharide (EPS) Because unpunfied endotoxin is of concern in the production ol pharmaceutical compounds (eg. proteins produced in t coli using iecombinant
  • teim endotoxin refers to unpunfied endotoxin
  • Bacterial endotoxin is a well known pvrogen
  • ⁇ s used herein the teim endotoxm-free when used in leterence to a composition to be admmisteicd parenterallv (with the exception of intrathecal administration) to a host means that the dose to be dehveied contains less than 5 F U/kg bodv weight [I DA Guidelines lor
  • compositions containing less than oi equal to than 25() endotoxin units (EU)/mg of purified recombinant protein are herein defined as "substantially endotoxm- free.”
  • the composition contains less than or equal to 100. and most preferably less than or equal to 60. (EU)/mg of purified recombinant protein
  • administration ot bacterial toxins or toxoids to adult humans for the purpose ol vaccination involves doses of about 10-500 ⁇ g protein/dose I herefore.
  • the L M test is accepted by the U S. FDA as a means of detecting bacterial endotox s (21 Cl R. ⁇ 660.100 -105). Studies have shown that the L ⁇ E test is equivalent oi superior to the USP rabbit pvrogen lest for the detection of endotoxin and thus the 1 Al. test can be used as a surrogate for pyrogenicity studies in animals [F C Perason. P ⁇ rogens endoioxins I AL testing and Marcel Dekker. New York (1985). pp 150-!55
  • m ⁇ novalent when used in reference to a clostridial vaccine reters to a vaccine which is capable ol provoking an immune response in a host animal duected against a single type of clostridial toxm
  • a host with ( ' hoiulinum type A toxin vaccine induces antibodies in the immunized host which protect against a challenge with t pe A toxin but not against challenge with ty e B. C D. E. F or G toxins.
  • the type A vaccine is said to be monovalent.
  • a "muluvale ⁇ t" vaccine provokes an immune response in a host animal directed against several (/ e .
  • clostridial toxins are said to be multivalent (in particular, this hypothetical vaccine is bivalent) ⁇ .s used heiein the term "lmmunogenicallv-effective amount" refers to that amount ot an immunogen required to invoke the production of protecti e levels ol antibodies m a host upon vaccination
  • the teim 'piotective level when used in reference to the le el of antibodies induced upon immunization of the host with an immunogen which comprises a bacterial toxin means a level of ciiculating antibodies sufficient to protect the host Irom challenge with a lethal dose of the toxin
  • Ihe teim leceptoi-binding domain when used in retcrencc to a ( botulinum toxin icteis to the caiboxv-teiminal portion ot the heavv chain (H ( or the C liagment) of the toxin which is piesumed to be icsponsible tor the binding ol the active toxin (/ c the derivative toxm comp ⁇ sing the H and I chains )o ⁇ ned via disullide bonds) to leceptois on the surface ol svnaptosomes
  • the receptor-binding domain tor C hoiulinum tvpe ⁇ toxin is defined heiem as compiismg amino acid lesidues 861 thiough 1296 ol SI Q ID NO 28
  • Ihe icceptoi- binding domain for ( hoiulinum tvpe B toxin is defined herein as comprising amino acid les
  • Ihe leceptor-binding domain ol C hoiulinum tvpe F toxin is defined herein as comprising ammo acid lesidues 835 thiough 1250 ot SF Q ID NO 50 (Beluga stiain)
  • Ihe leceptoi -binding domain ot C hoiulinum tvpe i toxin is defined herein as comprising ammo acid lesidues 853 through 1274 ot SEQ ID NO 71
  • Ihe receptor-binding domain ot ( botulinum tvpe CJ toxm is defined herein as comprising ammo acid residues 853 thiough 1297 ol STQ ID NO 77
  • the present invention contemplates fusion proteins comprising the receptor-binding domain of C. hoiulinum toxins from serotypes A-G including the variants found among different strains within a given serotype. I he receptor-binding domains listed above are used as the prototype for each strain within a serotype. Fusion proteins containing an analogous region from a strain other than the prototype strain are encompassed by the present invention.
  • F usion proteins comprising the receptor binding domain (i.e.. C fragment ) of botuhnal toxins may include amino acid residues located beyond the termini of the domains defined above.
  • the pFlisBotB protein contains amino acid residues 846- 1291 of SEQ ID O:40; this fusion protein thus comprises the receptor-binding domain for C. hoiulinum type
  • pHisBotE contains amino acid residues 827- 1 252 of SEQ I F) NO:50 and pHisBotG contains amino acid residues 85 1 - 1 297 of SEC) I D NO: 77.
  • both pHisBotE and pHisBotG fusion proteins contain a few amino acids located beyond the N-terminus of the defined receptor-binding domain.
  • the terms "native gene” or “native gene sequences” are used to indicate DNA sequences encoding a particular gene which contain the same DNA sequences as found in the gene as isolated from nature.
  • synthetic gene sequences are DNA sequences w hich are used to replace the naturally occurring DNA sequences w hen the naturally occurring sequences cause expression problems in a giv en host cell.
  • naturally- occurring DNA sequences encoding codons which are rarely used in a host cell may be replaced (e.g.. by site-directed mutagenesis) such that the synthetic DNA sequence represents a more frequently used codon.
  • the native DNA sequence and the synthetic DNA sequence w ill pref erably encode the same amino acid sequence.
  • the present invention relates to the production of polypeptides derived from toxins particularly in recombinant host cells.
  • the present invention provides a host cell containing a recombinant expression vector, said vector encoding a protein comprising at least a portion of a Closiridium hoiulinum toxin, said toxin selected from the group consisting o ⁇ type B toxin and type E toxin.
  • the present invention is not limited by the nature of sequences encoding portions of the ( ' . hoiulinum toxm.
  • sequences may be derived irom the native gene sequences or alternatively thev may comprise synthetic gene sequences Synthetic gene sequences aie emploved when expression of the native gene sequences is problematic in a given host cell (e g, when the native gene sequences contain sequences resembling yeast transcription termination signals and the desired host cell is a veast cell)
  • the host cell is capable of expressing the recombinant C hoiulinum toxin protein at a level gieatet than oi equal to 2% to 40% of the total cellular piotem and preleiably at a level gteater than or equal to 5% of the total cellular protein
  • the host cell is capable of expressing the recombinant ( hoiulinum toxm piotem as a soluble protein at a level greater than oi equal to 025% of the total cellulai piotem andêtrablv at a level greater than or equal to 025% to 10% ot the total cellular protein
  • Ihe present invention is not limited bv the natute ot the host cell emploved foi the pioduction ol iecombinant C hoiulinum toxm pioteins
  • the host cell is an / coli cell
  • the host cell is an insect cell, paiticulailv pieterred insect host cells are Spodopieia fiugipeida (Sf9) cells
  • the host cell is a veast cell particularlv pieterred veast cells are Pic/va pashms cells in anothei embodiment the invention provides a host cell containing a iecombinant expiession said vectoi encoding a fusion protein comprising a non-toxm protein sequence and at least a portion of a C losit idiuni hoiulinum toxm.
  • said toxin selected tiom the moup consisting ol tv e B toxm and t pe E toxin
  • the invention is not limited bv the nature ol the poition ol the C losntdium hoiulinum toxm selected In a prefeired embodiment the portion ol the toxm compiises the receptor binding domain (i e .
  • the non-toxm piotem sequence comprises a polv- histidmc tiact A number ot alternative fusion tags or lusion paitners are known to the art (c g MBP. GST protein A.
  • the piesent invention further provides a vaccine comprising a tusion protein said lusion protein comprising a non-toxm protein sequence and at least a portion ot a C losliidium hoiulinum toxin said toxin selected from the group consisting ot tvpe B toxm and tvpe I toxin Ihe vaccine mav be a monovalent vaccine (/ c containing onlv a toxin B tusion 5 piotem or a toxin E fusion protein) a bivalent vaccine (/ c containing both a toxin B fusion protein and a toxm E fusion protein) or a trivalent 01 higher vaiencv vaccine
  • the toxin B tusion protein and/oi toxin F tusion protein is combined with a lusion pi
  • the portion ot the toxin comprises the leceptor binding domain (/ c the C fiagme ⁇ t ot the toxin)
  • Ihe present invention is not limited bv the natuie ot the non-toxin protein sequence emploved
  • the non-toxin protein sequence comp ⁇ ses a polv-histidme ti act ⁇ number ol alternative lusion tags or lusion partneis are known to the ait (c _j MBP GSI
  • ->s Ihe present invention is not limited bv the method emploved for the generation of v ccine compiising fusion proteins comprising a non-toxm piotein sequence and at least a poition ol a C losliidium hoiulinum toxin
  • the tusion proteins mav be pioduced bv lecombmant DNA means using either native or svnthetic gene sequences expressed a host cell
  • Ihe piesent invention is not limited to the pioduction ot vaccines using recombinant host cells cell free in transc ⁇ ption/tianslation sv stems mav be emploved foi the
  • the fusion proteins of the present invention may be generated by sy nthetic means (i. e.. peptide synthesis).
  • the present invention further provides a method of generating antibody directed against a Closiridium hoiulinum toxin comprising: a) providing in any order: i ) an antigen comprising a fusion protein comprising a non-toxin protein sequence and at least a portion ol ' a Closiridium hoiulinum toxin, said toxin selected from the group consisting of type B toxin and ty pe E toxin, and ii ) a host: and b) immunizing the host w ith the antigen so as to generate an antibody .
  • the antigen used to immunize the host also contains a fusion protein comprising a non-toxin protein sequence and at least a portion of " Closiridium hoiulinum ty e A toxin.
  • a fusion protein comprising a non-toxin protein sequence and at least a portion of " Closiridium hoiulinum ty e A toxin.
  • the portion of the toxin comprises the receptor binding domain (i. e.. the C fragment of the toxin).
  • the present inv ention is not limited by the nature of the non-toxin protein sequence employed.
  • the non-toxin protein sequence comprises a poly-histidine tract.
  • a number o ⁇ alternative fusion tags or fusion partners arc know n to the art (e.g.. MBP. GST. protein A. etc. ) and may be employ ed for the generation of fusion proteins comprising v accines.
  • a fusion partner ⁇ i. e.. the non-toxin protein sequence
  • the fusion partner may be removed from the recombinant O hoiulinal toxin protein if desired ( i. e.. prior to administration of the protein to a subject ) using a variety of methods known to the art ⁇ e.g..
  • the present invention is not limited by the nature of the host employed for the production of the antibodies of the invention.
  • the host is a mammal, preferably a human.
  • the antibodies of the present invention may be generated using non-mammalian hosts such as birds, preferably chickens.
  • the method of the present invention further comprised the step c ) of collecting the antibodies from the host.
  • the method of the present invention further comprises the step d) of purifying the antibodies.
  • the present invention further provides antibodies raised according to the above methods.
  • the present inv ention further contemplates multivalent vaccines comprising at least two recombinant ' . hoiulinum toxin proteins derived from the group consisting of C. hoiulinum serotypes A. B. C I).
  • E. F. and G. Fhe invention contemplates bivalent, trivalent, quadrav alent. pent board.
  • heptavalent and septivalent vaccines comprising recombinant ( . hoiulinum toxin proteins.
  • the recombinant ' . hoiulinum toxin protein comprises the receptor binding domain (i.e.. C fragment) of the toxin.
  • DESCRIPTION OF THE INVENTION fhe present invention contemplates vaccinating humans and other animals with poly peptides derived from C. botulinum neuroloxins which are substantially endotoxin-free. These botuhnal peptides are also useful for the production of antitoxin. ⁇ nti-botulinal toxin antitoxin is useful for the treatment of patients effected by or at risk of symptoms due to the action of ( ' . hoiulinum toxins. T he organisms, toxins and individual steps of the present invention are described separately below .
  • a preferred embodiment of the method of the present inv ention is directed toward obtaining antibodies against Closiridium species, their toxins, enzymes or other metabolic by products, cell wall components, or synthetic or recombinant versions of any of these compounds. It is contemplated that these antibodies will be produced by immunization of humans or other animals. It is not intended that the present invention be limited to any particular toxin or any species of organism.
  • toxins from all Closiridium species are contemplated as immunogens. Examples of these toxins include the neuraminidase toxin of ( ' . buiyricum. C. sordellii toxins H I and FT. toxins A. B. C I). E. F .
  • toxins A. B and F oi C botulinum are contemplated as immunogens Table 2 above hsts various ( losliidium species, their toxins and some antigens associated with disease
  • antibodies directed against one toxin t g ( peifnngens tv e A enterotoxin) mav be used as an effective therapeutic against one or more tox ⁇ n(s) pioduced bv othei membeis of the genus Closiiidium or other toxin pioducmg oiganisms (c g Bacillus cams auiais iieptococais muians landobacia LLilcoacciicus Pseudomonas ua other Pseudomonas species etc ) It is lurthei contemplated that antibodies duected against the portion ot the toxin which binds to mammalian membianes (e g ( enterotoxin A) can also be used against othei oiganisms It is contemplated that these membrane binding domains
  • the method of the present invention contemplates immunizing non-mammals with bacterial tox ⁇ n(s) It is not intended that the piesent invention be limited to any particular toxm
  • toxm tiom all clostridial bacteria sources (see Table 2) aie contemplated as immunogens I xamples ot these loxms are C huiMiciim
  • ⁇ paiticularly prelerred embodiment involves the use ot baetenal toxin protein oi tiagments ol toxin proteins produced by molecular biological means (i e . iecombinant toxin proteins)
  • the immunogen comprises the leceptor-bmding domain
  • the immunogen comprises the leceptoi-bindmg domain ol C hoiulinum serotype B neurotoxin produced by recombinant DN ⁇ technology In vet anothei pretence!
  • the immunogen comprises the leceptoi-bmdmg domain legion ol ( hoiulinum serotype F " neurotoxin produced by recombinant DNA teehnologv
  • the immunogen comprises the leceptoi -binding domain icgion ol ( hoiulinum serotype C 1 neurotoxin pioduced by iecombinant DNA technology
  • the immunogen comprises the receptor-binding domain icgion of C botulinum serotype C2 neurotoxin produced by recombinant F)NA technology
  • the immunogen comprises the receptor-binding domain icgion ot C hoiulinum serotype D neurotoxin produced by recombinant DNA technology
  • the immunogen comprises the receptor-binding domain icgion ol ( ' hoiulinum serotype F neurotoxin pi
  • type B type C including C 1 and C2.
  • type F type E. and t pe J toxin hen immunization is used, the preferred non-mammal is liom the class -hrs All birds aie contemplated (e g duck, ostrich, emu. turkev. etc )
  • a preferred bird is a chicken impoitantiv chicken antibodv does not fix mammalian complement [See H N Benson et al . I Immunol 87616 (1961) ]
  • chicken ant ody will normally not cause a complement- dependent reaction [A A Benedict and K Yamaga.
  • the present invention contemplates all tvpes ol toxin modification, including chemical and heat treatment ot the toxin
  • the prefeired modification is lo ⁇ naldehv e tieatment
  • the present invention contemplates all modes ol immunization including subcutaneous, intramuscular, inlrapciitoneal. and intravenous or mtravasculai ii ⁇ ection as well as pei s administration ot immunogen I he present invention further contemplates immunization with or without adjuvant.
  • uvant is defined as a substance known to increase the immune response to other antigens w hen administered with other antigens.
  • uvant it is not intended that the present invention lie limited to any particular type of adjuvant — or that the same adiuvant. once used.
  • the present invention contemplates all types of ad
  • a chicken is administered tox ⁇ n( s) on day zero and subsequently receives tox ⁇ n(s) in intervals thereattei It is not intended that the present invention be limited by the particular interv als oi doses Similarly , it is not intended that the present inv ention be limited to any particular schedule loi collecting antibody I he pref ei red collection time is sometime after day 100.
  • chicken antibody produced in this manner can be buffei - extiaetcd and used analytically While unpunfied.
  • this preparation can serv e as a reference f or acliv itv ot the antibody prior to further manipulations ( e immunoal tlnity puri fication )
  • the present inv ention contemplates purif y ing to increase the ef fectiv eness ol both non-mammalian antitoxins and mammalian antitoxins Speciilcallv .
  • the present invention contemplates increasing the percent ot toxin-reactive immunoglobulin I he pref erred purification approach tor avian antibody is poly ethylene gly col ( PEG) sepaiation
  • Fhe present inv ention contemplates that avian antibody be initially purified using simple, inexpensive procedures
  • chicken antibody f rom eggs is punged by extraction and precipitation with PEG PEG purification exploits the differential solubility ot lipids (which are abundant in egg yolks) and yolk proteins in high concentrations of PEG 8000 [Poison et al . Immunol. Comm 9 495 ( 1980) J fhe technique is rapid, simple, and relativ ely inexpensive and y ields an immunoglobulin traction that is significantly purer in terms of contaminating non-immunoglobulin proteins than the comparable ammonium sulfate fractions of mammalian sera and horse antibodies.
  • PEG-purifled antibody is sufficiently pure that the present invention contemplates the use of PEG-purified antitoxins in the passive immunization of intoxicated humans and animals.
  • T he present invention contemplates antitoxin therapy for humans and other animals intoxicated by bacterial toxins.
  • a preferred method of treatment is by intravenous administration of anti-boutlinal antitoxin: oral administration is also contemplated for other clostridial antitoxins.
  • T he present invention contemplates significantly reducing side effects so that this balance is more easily achieved.
  • Treatment according to the present invention contemplates reducing side effects by using PEG-purified antitoxin from birds.
  • the treatment of the present invention contemplates the use of PEG-purified antitoxin from birds.
  • T use of yoik-derived.
  • PFXi-purif ed antibody as antitoxin allows for the administration of: 1 ) non( mammalian)-complemcnt-flxing.
  • avian antibody 2 ) a less heterogeneous mixture of non-immunoglobulin proteins; and 3 ) less total protein to deliver the equivalent weight of active antibody present in currently available antitoxins.
  • the non-mammalian source of the antitoxin makes it useful for treating patients w ho are sensitive to horse or other mammalian sera.
  • B Delivery Of Antitoxin
  • antitoxin is delivered in a solid form (e.g.. tablets).
  • antitoxin is delivered in an aqueous solution.
  • the solution has sufficient ionic strength to solubilize antibody protein, vet is made palatable for oral administration.
  • the delivery solution may also be buffered (e.g., carbonate buffer pH 9.5) which can neutralize siomach acids and stabilize the antibodies when the antibodies are administered orally.
  • the delivery solution is an aqueous solution.
  • the delivery solution is a nutritional formula.
  • the deliv ery solution is infant formula.
  • Yet another embodiment contemplates the delivery of lyophilized antibody encapsulated or microencapsulated inside acid-resistant compounds.
  • enteric coatings which are resistant to gastric fluid and whose release (i. e.. dissolution of the coating to release the pharmaceutical compound ) is pFl dependent are commercially available
  • Eudragit J* S is soluble in intestinal lluid from pl l 7.0; this coating can be used to microencapsulate ly ophilized antitoxin antibodies and the particles are suspended in a solution having a pl l above or below pl l 7.0 for oral administration. The microparticles will remain intact and undissolved until they reached the intestines where the intestinal pH would cause them to dissolve thereby releasing the antitoxin.
  • antitoxin is administered orally in either a delivery solution or in tablet form, in therapeutic dosage, to a subject intoxicated by the bacterial toxin which served as immunogen for the antitoxin.
  • I he invention also contemplates a method of treatment which can be administered proph aelically.
  • antitoxin is administered orally , in a delivery solution. in therapeutic dosage, to a subject, to prevent intoxication of the subject by the bacterial toxin which serv ed as immunogen for the production of antitoxin.
  • antitoxin is administered orally in solid form such as tablets or as microencapsulated particles. Microencapsulation o ⁇ ly ophilized antibody using compounds such as Eudragit® ( Rohm GmbH) or polyethylene glycol . which dissolve at a wide range of pH units, allows the oral administration of solid antitoxin in a liquid form (i c .
  • a suspension to recipients unable to toleiate administration of tablets (e g . children or patients on feeding tubes)
  • the subject is a child
  • antibody raised against whole baetenal oi ga sm is administered orally to a subject, in a dehvei y solution, in therapeutic dosage
  • I he inv ention contemplates the generation of mono- and multiv alent v accines lot the piotection of an animal (particularly humans) against seveial clostridial species Of particulai interest aie v accines which stimulate the production of a humoral immune response to ( hoiulinum ( i ei am and ⁇ diffic ile in humans T he antigens comprising the v accine prepai ation mav be nativ e or recombinantly produced toxin proteins f rom the clostridial species listed above When toxin proteins are used as immunogens thev are generally modified to reduce the toxicity This modification may be by chemical or genetic ( / e .
  • I n general genetic detoxification ( / e . the expression ol nontoxie ti agments in a host cell ) is preferred as the expression ol nontoxic fragments in a host cell piecludes thedecknce of intact, active toxin in the final preparation Howevei . w hen chemical modification is desired the prelerred toxin modification is lormaidehy de treatment
  • the recombinant C botulinum toxin proteins derived from serotpes A B and I may be used in conjunction w ith either recombinant or native toxins oi toxoids f i om othei sei oty pes ol ( botulinum.
  • a vaccine comprising C difficile and hoiulinum toxin pioteins ( native or i ecombinant or a mixture thereof) be used to stimulate an immune l espouse against C hoiulinum C lelani and ( difficile
  • the present invention further contemplates multi-valent vaccines comprising two or more botulinal toxin proteins selected from the group comprising recombinant C. botulinum toxin proteins derived from serotypes A. B. C (including C l and C2). D. E. F and G.
  • the adverse consequences of exposure to botulinal toxin would be avoided by immunization of subjects at risk of exposure to the toxin with nontoxic preparations which confer immunity such as chemically or genetically detoxified toxin.
  • Vaccines which confer immunity against one or more of the toxin types A. B. F : ,. F and G would be useful as a means of protecting humans from the deleterious effects of those ' hoiulinum toxins known to affect man. Indeed as the possibility exists that humans could be exposed to any of the seven serotypes of C. botulinum toxin (e.g.. during biological warfare or the production of toxin in a laboratory setting), multivalent vaccines capable of conferring immunity against toxin types A-G (including both C l and C2 toxins) would be useful for the protection of humans. Vaccines which confer immunity against one or more of the toxin types C D and E would be useful for veterinary applications.
  • Fhe botulinal neurotoxin is synthesized as a single polypeptide chain which is processed into a heavy ( H: - 100 kD) and a light (L: -50 kD) chain by cleavage w ith proteolvtic enzymes; these two chains are held together via disulfide bonds in the active toxin ( referred to as deriv ative toxin) [ B.R. DasGupta and I F Sugiyama. Biochem. Biophys. Res. C ' ommun. 48: 108 ( 1972): reviewed in B.R. DasGupta. J. Physiol. 84:220 ( 1990). H . Sugiyama. Microbiol. Rev. 44:419 ( 1980) and CL.
  • the heavy chain of the active toxin is cleaved by trypsin to produce two fragments termed 1 1, (also referred to as H , or C) and 1 1 N (also referred to as I E or B).
  • I he H fragment ( -46 kD) comprises the carboxy end of the 1 1 chain.
  • the H , fragment (-49 kD) comprises the animo end and remains attached to the L chain ( H L).
  • H L Neither H or H N L is toxic.
  • H c competes w ith whole derivative toxin for binding to synaptosomes and therefore 1 I ( is said to contain the receptor binding site.
  • Fhe H and H N fragments of botulinal toxin are analogous to the fragments C and B of tetanus toxin which are produced by papain cleavage.
  • Fhe C fragment of tetanus toxin has been shown to be responsible for the binding of tetanus toxin to purified gangliosides and neuronal cells ( Halpern and Loftus. J. Biol. Chem. 288: 1 1 1 88 ( 1993)].
  • Antisera raised against purified preparations of isolated botulinal IT and L chains have been shown to protect mice against the lethal effects of the toxin; however, the effectiveness of the two antisera differ with the anti-H sera being more potent ( I I. Sugiyama. supra).
  • C hoiulinum toxins isolated from different strains within a given serotype.
  • the C hoiulinum toxins contain about 1250-1300 amino acid residues.
  • C D and E toxins averages between 50 and 60% identity with a greater degree of homology being found between H chain-encoding regions than between those encoding L chains [Whelan el al (1992) Appl Environ.
  • the degree of identity between C hoiulinum toxins on the amino acid level reflects the level of DNA sequence homology
  • the most divergent area of DNA and ammo acid sequence is found within the carboxy-terminal area of the various (' hoiulinum H chain genes.
  • Fhis portion of the toxin i e.. H ( or the C fragment) plays a ma
  • toxin from different serotypes is thought to bind to distinct cell receptor molecules, it is not surprising that the toxins diverge significantly over this region
  • the present invention contemplates fusion proteins comprising portions ol C hoiulinum toxins from serotypes A-G including the variants found among different strains within a given serotype.
  • the present invention provides oligonucleotide pinners which may be used to amplify the C fragment or receptor-binding region of the toxin gene Irom various strains ol C hoiulinum serotype A. serotype B. serotype C (Cl and C2). serotype 1). serotype E.
  • serotype I and serotype G A large number of different strains of C hoiulinum serotype A. serotype B. serotype C serotype D serotype E and serotype F are available from the American Type Culture Collection (ATCC; Rockville. MD).
  • AICC provides the following. Ivpe A strains: 174 (ATCC 3502).457 (ATCC 17862). and NCTC 7272 (ATCC 19397).
  • Type C strains 2220 (ATCC 17782).2239 (ATCC 1778.3).2223 (A FCC 17784; a type C-[ ⁇ strain. C ' - ⁇ stiains produce C2 toxin).662 (ATCC 17849; a type C- ⁇ strain; C- ⁇ strains produce mainly Cl toxin and a small amount of C2 toxin).2021 (ATCC 17850: a type C- ⁇
  • VPI 3803 JJ strain and VPI 3803 (ATCC 25766); I ype D strains. ATCC 9633.2023 (ATCC 17851), and VPI 5995 (ATCC 27517); Type L strains. ATCC 43181.36208 (ATCC 9564).2231 (ATCC 17786).2229 (ATCC 17852).2279 (ATCC 17854) and 2285 (ATCC 17855) and Type F strains 202F (ATCC 23387).
  • VPI 4404 (ATCC 25764).
  • VPI 2382 AICC 27321) and Langeland (ATCC 35415)
  • Type G strain. 113/30 may be obtained from the
  • NCFB National Collection of Tood Bacteria
  • botuhnal toxins e g. types A-G
  • soluble recombinant fusion proteins e.g. types A-G
  • the C botulinum and C tetanus toxin proteins have similar structures [reviewed in F I Schantz and E A Johnson. Microbiol Rev 5680 (1992)]
  • the carboxy-terminal 50 kD tiagment of the tetanus toxin heavy chain (fragment C) is released by papain cleavage and has been shown to be non-toxic and immunogenic Recombinant tetanus toxin fragment C has been developed as a candidate vaccine antigen [A J Makoff et al .
  • Recombinant tetanus tiagment C has been expressed in L coli (A I Makoff et al . Bio/ lechnology. sup/a and Nucleic Acids Res 1710191 (1989). J I Halpern et al . Infect Immun 581004 (1990)j. yeast [MA Romanos el al . Nucleic Acids Res 191461 (1991)] and baculovirus [I G Charles et al . Infect Immun 591627 (1991)] Synthetic tetanus toxin genes had to be constructed to facilitate expression in veast (M A Romanos el al . supra) and L coli [ ⁇ I Makoff ei at .
  • botulinum type A toxin C fragment/MBP fusion protein was not shown to be substantially free of endotoxin contamination.
  • Experience with recombinant C. botulinum type A toxin C fragment/MBP fusion proteins shows that the presence of the MBP on the fusion protein greatly complicates the removal of endotoxin from preparations of the recombinant fusion protein (.se Ex. 24. infra).
  • Expression of a synthetic gene encoding C. botulinum type A toxin C fragment as a soluble protein excreted from insect cells has been reported [Middlebrook and Brown ( 1995). supra]: no details regarding the level of expression achieved or the presence of endotoxin or other pyrogens were provided.
  • immunization with the recombinant protein produced in insect cells was reported to protect mice from challenge with C. botulinum toxin A.
  • Inclusion body protein must be solubihzed prior to purification and/or administration to a host. The harsh treatment of inclusion body protein needed to accomplish this solubilization may reduce the immunogenicity of the purified protein.
  • recombinant proteins to be used as vaccines are expressed as soluble proteins at high levels (i. e.. greater than or equal to about 0.75% of total cellular protein) in E. coli or other host cells (e.g.. yeast, insect cells, etc.). T his facilitates the production and isolation of sufficient quantities of the immunogen in a highly purified form (i. e.. substantially free of endotoxin or other pvrogen contamination).
  • the ability to express recombinant toxin proteins as soluble proteins in E. coli is advantageous due to the low cost of growth compared to insect or mammalian tissue culture cells.
  • the ( ' . hoiulinum type B neurotoxin gene has been cloned and sequenced from two strains o ⁇ C hoiulinum type B [Whelan el al. ( 1992) Appl. Environ. Microbiol. 58:2345 ( Danish strain) and Hutson et al. ( 1994) Curr. Microbiol. 28: 101 ( Eklund 1 7B strain)].
  • T he nucleotide sequence of the toxin gene derived from the Eklund 1 7B strain ( ATCC 25765) is available from the EMBL/GenBank sequence data banks under the accession number X71 43: the nucleotide sequence of the coding region is listed in SEQ ID NO:39. The amino acid sequence of the C.
  • botulinum type B neurotoxin derived from the strain Eklund 1 7B is listed in SEQ I D NO:40.
  • the nucleotide sequence of the ( ' . botulinum serotype B toxin gene derived from the Danish strain is listed in SEQ ID NO:41 .
  • the amino acid sequence of the C. hoiulinum type B neurotoxin derived from the Danish strain is listed in SEQ ID NO:42.
  • the C hoiulinum type B neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a di er composed of a light and a heavy chain linked via disulfide bonds
  • the light chain is responsible for pharmacological activitv (/ e .
  • the N-terminal portion ot the heavy chain is thought to mediate channel formation while the C-terminal portion mediates toxm binding, the type B neurotoxin has been reported to exist as a mixture of predominantly single chain with some double chain (Whelan et al supia) T he 50 kD carboxv -terminal portion ot the heav y chain is referred to as the C tiagment or the l l t domain
  • the present invention reports for the first time, the expression of the C fragment of C botulinum tvpe B toxm in heterologous hosts (e g E coli)
  • the C hoiulinum ty pe T neurotoxin gene has been cloned and sequenced from a numbei ot dif ferent strains [ Poulet el al ( 1992) Biochem Biophys Res Commun 1 83 107 W helan et al ( 1992) 1 ui J Biochem 204 657. and Fuj ⁇ el al ( 1993) I Gen Microbiol 1 9 79
  • I he nucleotide sequence of the type E toxin gene is available from the EMBL sequence data bank under accession numbers X62089 (strain Beluga) and X62683 (strain NC I C 1 1 2 1 9).
  • the nucleotide sequence ot the coding region (strain Beluga) is listed in SEQ ID NO 45
  • Hie ammo acid sequence ot the ( hoiulinum ty pe E neurotoxin deriv ed from spain Beluga is listed in SEQ I D NO 46
  • the ty pe F neurotoxin gene is s y nthesized as a single poly peptide chain which may be converted to a double-chain form (i c . a heav v chain and a light chain) by cleav age w ith trypsm. unlike the ty pe A neurotoxin.
  • the tvpe F euiotoxin exists essentially onlv in the single-chain form
  • the 50 kD cai boxv -terminal poi tion ot the heavy chain is referred to as the C f ragment oi the I I, domain
  • I he botulinum ty pe C l u. Y and G neurotoxin genes have been cloned and sequenced
  • the nucleotide and amino acid sequences of these genes and toxins are provided heie I he invention prov ides methods lor the expression of the C fragment from each of these toxin genes in heterologous hosts and the purification of the resulting iecombinant pi oteins
  • ect invention provides methods which allow the production ot soluble C holulinuin toxin proteins in economical host cells (e g E coli)
  • economical host cells e g E coli
  • the subject inv ention prov ides methods which allow the production ot soluble botuhnal toxin proteins in v east and insect cells I uither methods lor the isolation of purified soluble C hoiulinum
  • C botulinum toxin proteins which are suitable for immunization of humans and other animals are provided. These soluble, purified preparations of C botulinum toxin proteins provide the basis for improved vaccine preparations and facilitate the production of antitoxin
  • recombinant clostridial toxin proteins produced in gram-negative bacteria e g E coli
  • they are purified to remove endotoxin prior to administration to a host animal
  • an lmmunogcnically-effective amount of purified substantially endotoxin-free recombinant clostridial toxin protein is administered in anv ot a numbei ot physiologically acceptable carriers known to the ai t
  • the purified substantially endotoxin-li ee recombinant clostridial toxin protem mav be used alone or in conjunction with known adjutants, including potassium alum, aluminum phosphate, aluminum hydroxide.
  • uvant GmDP. C C Biotech Corp
  • R1BI adjuv ant MPL, R1BI Immunochemical Research. I no ).
  • QS21 Chembridge Biotech
  • the alum and aluminum-based adjutants are particularly preferred when vaccines arc to be administered to humans, however, any adjuvant approved tor use in humans mav be employed I he route ot immunization may be nasal, oral, intramuscular, lntrape ⁇ toneal or subcutaneous
  • the v accine comprises the C fragment ot eithei the C hoiulinum type A. B. C D. E. I . or G toxm and a polv-histidme tract (also called a histidine tag).
  • a f usion protem comprising the histidine tagged C fragment is expressed using the pi T senes ol expression vectors ( Nov agen)
  • the pF F expression sy stem utilizes a vector containing the TI promoter which encodes the fusion protein and a host cell which can be induced to express the 1 7 DNA polymerase ( i e .
  • C Iragment fusion proteins containing a histidine tract is not limited to the use ot a particulai expression vector and host strain Seveial commercially available expression vectors and host strains can be used lo express the C fragment protem sequences as a tusion protein containing a histidine tract (I or example, the pQE series (pQE-8. 12, 16. 17. 18. 30, 3 1. 32. 40. 41. 42. 50. 5 1. 52. 60 and 70) ot expression v ectors (Qiagen) which are used with the host strains M I 5fpRE P4] (Qiagen ) and
  • (Qiagen) can be used to express lusion proteins containing six histidine residues at the amino-terminus of the fusion protem)
  • I urthermore a number ot commercially available expression vectors which provide a histidine tract also provide a protease cleavage site betw een the histidine tract and the protein of interest (e . botulinal toxin sequences) Cleavage of the resulting fusion protein with the appropriate protease will remove the histidine tag from the protein of interest (e g , botulinal toxin sequences) (see Example 28a. infra) Removal of the histidine tag may be desirable prior to administration of the i ecombinant botuhnal toxin protem to a subject (e g . a human)
  • sample in the pi esent specification and claims is used in its broadest sense On the one hand it is meant to include a specimen or cultuie On the other hand, it is meant to include both biological and env ironmental samples
  • Biological samples may be animal, including human, fluid, solid ( . stool ) oi tissue, liquid and solid food products and ingredients such as dairy items, v egetables, meat and meat by -pi oducis. and waste Env ironmental samples include env ironmental material such as sin face matter soil, water and industrial samples, as well as samples obtained Irom food and dairy processing instruments, apparatus, equipment, disposable and non-disposable items I hese examples at e not to be consti ued as limiting the sample ty pes applicable to the present inv ention
  • I he invention contemplates detecting bacterial toxin by a competitiv e immunoassav method that utilizes recombinant toxin A and toxin B proteins, antibodies raised against i ecombinant bacterial toxin proteins
  • a fixed amount ot the recombinant toxm proteins are immobi l ized to a solid support (c g a microtitcr plate) follow ed bv the addition of a biological sample suspected of containing a bacterial toxin I he biological sample is first mixed w ith al Unity -purified oi PEG fractionated antibodies directed against the recombinant toxm pi otem
  • a l eporter reagent is then added which is capable ot detecting the presence ot antibody bound lo the immobilized toxm protein
  • the reporter substance may comprise an antibody w ith binding specificity tor the antitoxin attached to a molecule which is used to identify the presence of the repo
  • Affinity-purified antibodies directed against recombinant bacterial toxin proteins are immobilized to a solid support (e.g.. microtitcr plates). Biological samples suspected of containing bacterial toxins are then added followed by a washing step to remove substantially all unbound antitoxin, fhe biological sample is next exposed to the reporter substance, which binds to antitoxin and is then washed free of substantially all unbound reporter substance.
  • the reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used lo identify the presence of the reporter substance. Identi fication of the reporter substance in the biological tissue indicates the presence of the bacterial toxin.
  • bacterial toxin be detected by pouring liquids (e.g.. soups and other fluid foods and feeds including nutritional supplements for hu ans and other animals ) over immobilized antibody which is directed against the bacterial toxin.
  • liquids e.g.. soups and other fluid foods and feeds including nutritional supplements for hu ans and other animals
  • immobilized antibody will be present in or on such supports as cartridges, columns, beads, or any other solid support medium.
  • unbound toxin is substantiall remov ed by washing. Fhe exposure of the liquid is then exposed to a reporter substance w hich detects the presence of bound toxin.
  • the reporter substance is an enzyme.
  • Iluorescent dye is an enzyme.
  • an antibod w hich is directed against the toxin ( i.e.. in a "sandwich” immunoassay ).
  • I t is also contemplated that the detection sy stem will be developed as necessary (e.g.. the addition of enzyme substrate in enzyme sy stems: observation using Iluorescent light for fluorescent dye sy stems: and quantitation of radioactivity for radioactive systems).
  • HC1 hydroochloric acid
  • 0() lethal dose lor 100% of experimental animals
  • aa amino acid
  • HPLC high performance liquid chromatography
  • kD kilodaltons
  • gm grams
  • ⁇ g micrograms
  • mg milligrams
  • ng nanogiams
  • ⁇ l microhters
  • mi mi (milhhters); mm (tmlhmeteis).
  • nm nanometers
  • M molar
  • mM moilhmolar
  • MW moeeulai weight
  • sec seconds
  • CiA hen a recombinant piotem is described in the specification it is referred to in a short-hand manner by the ammo acids the toxin sequence piesent in the recombinant prote rounded to the nearest 10 I or example, the recombinant protein pMB 1850-2360 contains amino acids 1852 through 2362 ot the C difficile tox B protein
  • the specification gives detailed construction details for all recombinant proteins such that one skilled in the ail will know precisely which ammo acids are present in a given recombinant protein
  • Ihe example involved (a) pieparation ol the baetenal immunogen (b) immunization (c) purification ot anti-bacterial chicken antibodies and (d) detection of anti-bactenai antibodies in the purified IgY prepaiations
  • the ii ] suspensions contained approximately 3 x 10 organisms/ml. and the "7 suspensions contained approximately 2 x 10 ' ' organisms/ml. [ Id. ] ' Fhe four resulting concentration-adjusted suspensions of formalin-treated C. difficile organisms were considered to be "bacterial immunogen suspensions. " These suspensions w ere used immediately after preparation for the initial immunization. [See section (b). [
  • T he formalin-treatment procedure did not result in 100% non-viable bacteria in the immunogen suspensions.
  • the formalin concentration and length of treatment were both increased f r subsequent immunogen preparations, as described below in T able 3. (Although viability was decreased with the stronger formalin treatment. 100% inviability of the bacterial immunogen suspensions was not reached. )
  • the formalin solutions were prepared in normal saline instead of PBS. At day 49. the day of the fifth immunization, the excess volumes of the four previous bacterial immunogen suspensions were stored frozen at -70°C for use during all subsequent immunizations.
  • C difficile strains described above were thawed and diluted to a concentration of approximately 1 x 10 7 organisms/ml using PBS. pll 7.2. In this way. two separate coating suspensions were prepared, one for each immunizing strain Into the wells ot 96-vvell microtiter plates (falcon. Pro-Bind Assay Plates) were placed 100 ⁇ l volumes ol the coating suspensions. In this manner, each plate well received a total ot approximatelv 1 ⁇ 10" organisms ol one strain or the other. The plates were then incubated at 4°C overnight The next morning, the coating suspensions were decanted, and all wells were washed three times using PBS In order to block non-specific binding sites.
  • pll 9.5 for the PBS in the final wash fhe plates were developed bv the addition of 100 ⁇ l of a solution containing I mg/ml para-nitrophenyl phosphate (Sigma) dissolved in 5() m Na-X ' O-. 10 mM MgC pll 95 u> each well, and incubating the plates at room temperature in the dark tor 45 minutes Ihe absorbance ol each well was measured at 410 nm using a Dynatech MR 700 plate reader In this mannei.
  • each ol the four IgY preparations described above was tested for reactivity against both ot the immunizing ( ' difficile strains: strain-specific, as well as cross-reactive activity was determined fable 5 shows the results of the whole-organism ELISA. All four IgY preparations demonstrated significant levels of activity, to a dilution of 1 62.500 or greater against both of the immunizing organism strains 1 herefore.
  • mice were oiallv admmisteied 30 mg clmdamvcin HCI (Sigma) in I ml of atei This ug predisposes hamsteis to ( difficile infection bv altering the normal intestinal flora On dav two the animals were given 1 ml ot the preimmune lgY/Ensure « formula at time zero.2 hours 6 hours and 10 hours At 1 hour on dav two different groups ot animals were inoculated oiallv with saline (control) oi 10 ⁇ ⁇ ( ⁇ 10" oi 10* L difficile oiganisms in I ml of saline I rom davs 3-12 animals were given I ml of the preimmune lg /I nsu ⁇ e' ⁇ foimula ihiee times daily and observed tor the onset of diarrhea and death L aeh animal was housed in individual cage and was ottered lood and watei a
  • Ihe cxpeiiment in (a) was lepeated using three groups ol seven hamsteis each Gioup A icceived no chndumvcin or difficile and was the survival contiol Gioup B received chndamvem.
  • Group C received clindamycin. 10 " C difficile organisms, and hyperimmune anti-C difficile IgY on the same schedule as Group B.
  • the anti-C. difficile IgY was prepared as described in E.xample 1 except that the 12%) PEG preparation w as dissolved in one fourth the original yolk volume of Ensured.
  • Hamsters in the control group A did not develop diarrhea and remained healthy during the experimental period.
  • Hamsters in groups B and C developed diarrheal disease.
  • Anti-C difficile IgY did not protect the animals from diarrhea or death, all animals succumbed in the same time interval as the animals treated with preimmune IgY.
  • w hile immunization with w hole organisms apparently' can improve sub-lethal sy mptoms with particular bacteria (see U.S. Patent No. 5.080.895 to I I. Tokoro). such an approach does not prov e to be productive to protect against the lethal effects of C. difficile.
  • Example 1 otal volk immunoglobulin (IgY) was extracted as described in Example 1(e) and the 0 IL pellet was dissolved in the onginal volk volume ol PBS ith thimeiosa!
  • pll 95 Blots were developed in lreshlv prepared alkaline phosphatase substrate butlei (100 ⁇ g/ml nitroblue tetrazohum (Sigma).50 ⁇ g'ml 5-b ⁇ omo-4- chloro-3-mdolv phosphate (Sigma).5 mM MgCU in 50 mM Na-,C () ,.
  • Ihe Western blots are shown in 1 igure 1 Ihe anli-C hoiulinum IgY reacted to the toxoid to give a broad immunoreactive band at about 145-150 kD on the reducing gel I his toxoid is relractive to disulfide cleavage by reducing agents due to lormalm ciosslmkmg Ihe immune IgY reacted with the active toxin complex, a 97 kD ( hoiulinum ivpe A heavy chain and a 53 kD light chain The preimmune IgY was unreactive to the ( hoiulinum complex oi toxoid m the Western blot c) Antitoxin Antibodv Titer
  • the IgY antibodv titer to C botulinum tvpe A toxoid of eggs harvested between dav 409 and 423 was evaluated bv I LISA prepared as follows Ninetv-six-well falcon Pro-bind plates eie coated overnight at 4°C with 100 ⁇ l/well toxoid [B R Singh &.
  • IgY In ordei to investigate the most effective delivery formula for IgY. e used IgY which was raised against ( loialus dunssus lerrificus venom Three eggs were collected fiom hens immunized with the C dunssus teiiificus venom and IgY was extracted tiom the yolks using the modified Poison piocedure described bv Thallev and ( anoll
  • the egg yolks were separated trom the whites, pooled and blended with four volumes ot PBS Powdered PEG 8000 was added to a concentiation of 35% fhe mixture was eentiifuged at 10.000 ipm toi 10 minutes to pellet the pi capitated protein, and the supernatant was filtered thiough cheesecloth to remove the lipid lavei Powdered PI G 8000 was added to the supernatant to bung the final PFG concentration to 12% (assuming a PEG concentration ol 35% in the supernatant) Ihe 12% PEG/IgY ' mixtute was divided into two equal volumes and centrifuged to pellet the IgY
  • PBS I he species specific secondary antibodv (goat anti-chicken immunoglobulin alkalme-phosphatase conjugate (Sigma) was diluted 1 .750 in PBS containing I mg/ml BS A and added to each wel l of the microtiter plate After 2 hours of incubation at l oom temperature, the unbound secondary antibody was removed by w ashing the plate as betore. and freshly prepared alkaline phosphatase substrate ( Sigma) al 1 mg/ml in 50 mM NaX ' O-. 10 mM MgCE. pl l 9.5 was added to each well, fhe color development was measured on a Dynatech MR 700 microplatc reader using a 412 nm filter f he results are show n in fable 8.
  • Ihe IgY' preparations used in this example are the same PBS-d ⁇ ssolved/I EC) dialvzed antivenom materials obtained in Example 4 above, mixed with an equal volume of Enfam ⁇ f ⁇ I wo mice were used in this experiment, each receiving a different diet as lollows
  • mice were given the corresponding mixture as their onlv source ot lood and water.
  • mice had ingested their respective tluids.
  • each tube was refilled with approximately 10 ml of the appropriate fluid first thing in the moin g Bv mid-mornmg there was about 4 lo 5 ml of liquid left in each lube
  • stool samples were collected tiom each mouse, weighed, and dissolved in approximatel y 500 ⁇ l PBS per 100 mg stool sample.
  • One hundred and sixty mg of control stools (no antibodv) and 99 mg of experimental stools (specific antibody) in 15 ml microfuge tubes were dissolved in 800 and 500 ⁇ l PBS. respectively.
  • I here was some active antibodv in the fecal extract fi om the mouse giv en the specific antibodv in 1 ntam ⁇ l ⁇ formula, but it was present at a v ery low lev el. Since the samples were assav ed at an initial 1 .5 dilution, the binding observed could have been higher w ith less dilute samples ( onseqiienth . the mice were allowed to continue ingesting either regular food and water oi the specific IgY' in EnfamiHi formula, as appropriate, so the assay could be repeated Another I 1 ISA plate was coated ov ernight with 5 ⁇ g/ml of C el t v enom m PBS
  • hoiulinum toxin type A complex obtained from Eric Johnson ( Universit y ' O ⁇ Wisconsin. Madison ) was 250 ⁇ g/ml in 50 mM sodium citrate, pl l 5.5. specific toxicity 3 1 0 ' mouse EDminister,/mg w ith parenterai administration. Approximately 40-50 ng/gm body weight was usuallv fatal w ithin 48 hours in mice maintained on conventional food and water.
  • mice were given 250 ⁇ l each of a preimmune IgY -f nsuie ⁇ mixture (preimmune IgY dissolved in 1/4 original volk volume) I hour befoie and I 2 hour and 5 houis alter administering botuhnal tox loxin concentrations given orallv langcd Irom approximately 12 to 312 ng/gm bodv weight (03 to 75 ⁇ g per mouse)
  • mice 10 per group were each given orallv a single dose of 1 ug each ol botuhnal tox complex in 100 ⁇ l ot 50 mM sodium citiatc pH 55 [he mice leceived 250 ⁇ l ticatments ot a mixture ot cither preimmune oi immune IgY m I nsuien (14 onginul volk volume) I hour betore and 1 '1 hour 4 hours and 8 hours alter botulinal toxm admmistiation The mice received three treatments pei dav toi two moie davs fhe mice were observed lor 96 hours Ihe survival and mortality are shown in [able 11
  • These mice eventually died 5 and 6 davs later
  • I ' oxin A is a potent cytotoxin secreted by pathogenic strains of C difficile, that plays a direct role damaging gastrointestinal tissues In more severe cases of ( ' difficile intoxication, pseudomembranous colitis can develop which may be fatal f his wouid be prevented bv neutralizing the effects of this toxm in the gastrointestinal tract
  • antibodies were produced against a portion of the toxm
  • the example involved (a) con
  • Serum Albumin Ihe synthetic peptide CQ flDGKKYYJ ' N-NIE was prepared commercially (Multiple Peptide Systems. San Diego. CA) and validated lo be 80% pure bv high-pressuie liquid chromatograph ⁇
  • the eleven amino acids lollowmg the evsteine icsidue represent a consensus sequence of a repeated amino acid sequence lound l xin ⁇ [Wren cl al . Infect Immun..59.3151-3155 (1991)
  • hens were each initially immunized on day zero by injection into two subcutaneous and two intramuscular sites with 1 mg of peptide conjugate that was emulsified in CTA ( GI BCO). The hens were boosted on day 14 and day 21 with 1 mg of peptide conjugate emulsified in I EA ( GIBCO).
  • IgY ' was purified from two eggs obtained before immunization ( pre-immune) and two eggs obtained 3 1 and 32 days after the initial immunization using PEG fractionation as described in Example 1 .
  • the wells were blocked for 2 hours at room temperature with 150 ⁇ l of a solution containing 5% ( v/v) Carnation* nonfat d v- milk and 1 % PEG 8000 in PBS. pl l 7.2. After incubation for 2 hours at room temperature, the wells were washed, secondary rabbit anti-chicken IgG-alkaline phosphatase ( 1 :750 ) added, the wells washed again and the color development obtained as described in E.xample 1 . The results are shown in Table 12.
  • Example involved (a) preparation ol the toxin immunogens. (b) immunization, (e) purification of the antitoxins, and (d) assay ot toxin neutralization activity
  • CTB This group was designated "CTB.” for the third immunization group.
  • 0.2 mi of toxoid A was first mixed with 0.2 ml of toxoid B. and the resulting mixture was emulsified in 0.4 ml of Tiler Max adjuvant.
  • This group was designated "CTAB.”
  • CTAB Tiler Max adjuvant.
  • three separate immunogen emulsions were prepared, with each emulsion containing a final concentration of 2.0 mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/ml toxoid A and 2.0 mg/ml toxoid B (CTAB).
  • White Leghorn hens obtained from a local breeder, were immunized as follow s: Group CTA. four hens were immunized, with each hen receiving 200 ⁇ g of toxoid A. v ia two intramuscular (l .M.) injections of 50 ⁇ l of CTA emulsion in the breast area.
  • Group CTB One hen was immunized with 200 ⁇ g of toxoid B. via two l.M. injections of 50 ⁇ l of CTB emulsion in the breast area.
  • Croup CTAB four hens were immunized, with each hen receiving a mixture containing 200 ⁇ g of toxoid A and 200 ⁇ g of toxoid B. via two l . M . injections of l OO ⁇ l of CTAB emulsion in the breast area.
  • the second immunization was
  • a toxin mixture 1 ml of a toxin mixture was prepared which contained 2()0 ⁇ g of native toxin A and 200 ⁇ g of native toxin B. T his toxin mixture was then emulsified in 0. 1 ml of filer Max adjuvant. A single CT AB hen was then immunized with the resulting immunogen emulsion, v ia two I.M. injections of l OOul each, into the breast area.
  • I ggs were collected from the hen in group CTB 10- 1 2 days follow mg the second immunization w ith toxoid ( day 35 immunization described in section ( b) abov e ), and Irom the hens in groups CT A and CTAB 20-2 1 days following the second immunization w ith toxoid l o be used as a pre-immune ( negative) control, eggs were also collected from unimmunized hens l i om the same flock.
  • Egg yolk immunoglobulin ( IgY 7 ) w as extracted Irom the 4 gioups ol eggs as described in Example 1 (c ).
  • T he toxin neutralization activity of the IgY solutions prepared in section (c ) above was determined using an assav sy stem that was modified from published methods
  • affinity-purified goat anti-C difficile toxm A ( l ech I ab) and affinity - purified goat anti-C difficile toxin B ( ech Lab) were also assayed for toxin neutralization activity .
  • the IgY solutions and goat antibodies were serially diluted using F 12 medium (GIBCO) which was supplemented with 2% FCS (GIBCO)(this solution will be referred to as " medium " for the remainder of this E.xample).
  • the resulting antibody solutions were then mixed with a standardized concentration of either native ( ' . difficile toxin A (Tech Lab), or native ( ' difficile toxin B (Tech Lab), at the concentrations indicated below . Following incubation at 37°C for 60 min..
  • toxin ⁇ - antibody mixtures were added lo the wells of 96-well microtiter plates (Falcon Microtest I II ) which contained 2.5 x 10 J Chinese Hamster Ovary (CHO) cells per well (the CT IO cells w ere plated on the previous day to allo them lo adhere to the plate wells).
  • the final concentration of toxin, or dilution of antibodv indicated below refers to the final test concentration of each reagent present in the respectiv e microtiter plate wells.
  • Toxin reference wells were prepared which contained CHO cells and toxin A or toxin B at the same concentration used for the toxin plus antibody mixtures ( these wells contained no antibody).
  • this Example utilized spectrophotometric methods to quantitate the ( ' . difficile toxin bioassav .
  • toxin A neutralizing activity of the CTA.
  • CTAB. and pre-immune IgY preparations, as well as the affinity-purified goat antitoxin A control dilutions of these antibodies were reacted against a 0. 1 ⁇ g/ml concentration of native loxin A (this is the approx. 50% cytotoxic dose of toxin A in this assay sy stem). The results are show n in Figure 3.
  • Ihe toxin B neutralizing activity of the CTAB and pie-immune IgY preparations and also the affinity -purified goat antitoxin B control was determined bv reacting dilutions ot these antibodies against a concentration ot native toxin B ol 01 ng/ml (approximatelv the 50% cvtotoxic dose ol toxin B in the assay system) Ihe tesults aie shown in 1 iguie 4
  • IgY prepaiations was determined bv leading dilutions ot these antibodies against a native loxin B concenlration ot 01 ⁇ g/ml (approximatelv 100% cvtotoxic dose ol toxm B in this assav system) Ihe results aie shown in I igure s
  • Example 8 fhe ability of the avian ( ' . difficile antitoxins, described in Example 8 above, to treat an established ( ' difficile infection w as evaluated using the Golden Syrian hamster model.
  • the Example involved (a) preparation of the avian C. difficile antitoxins, (b) in vivo treatment of " hamsters with established ( ' difficile infection, and (c ) histologic evaluation of cecal tissue.
  • Eggs were collected from hens in group CTAB described in Example 8 ( b) above. which were immunized with ( ' . difficile toxoids and native toxins A and B. Eggs purchased from a local supermarket w ere used as a pre-immune ( negative) control. Egg y olk immunoglobulin ( IgY ) was extracted from the 2 groups of eggs as described in Example 1 (e ). and the final IgY pellets were solubihzed in one-fourth the original yolk volume of Ensure 1 .'* nutritional formula.
  • IgY Egg y olk immunoglobulin
  • the avian ( '. difficile antitoxins prepared in section ( a ) above were evaluated for the ability to treat established ( ' . difficile infection in hamsters using an animal model system which was modified from the procedure which was described for the hamster protection study in Example 8(b) above
  • diffic ile inoculum, w hich contained approximately 1 0 C diffic ile strain 43596 organisms in ster ile saline ( difficile strain 43596 was chosen because it is representative ol one of the most ti equentlv -occurnng serogroups isolated from patients with antibiotic-associated pseudomembranous colitis [Delmee et al . .1 Clin. Microbiol . 28.2210-2214 ( 1990) ]
  • this w as the same ( diffic ile strain used in all of the previous [ xamples abov e, it w as again used in ol der to prov ide experimental continuity
  • treatment w as started f oi tw o of the tour groups ol animals
  • Each animal of one group was orally administered I 0 ml of the CTAB IgY ' preparation (prepaicd in section (a) above) at the following timepoints 0 hrs . 4 hrs .
  • mice and 8 hi s I he animals in this group were designated "CT AB-24"
  • the animals in the second group were each orally administered 1 0 ml of the pre-immune IgY preparation (also piepai ed in section ( a ) above ) at the same timepoints as f oi the C I ⁇ B gr oup 1 hese animals were e designated "P ⁇ e-24 " None was done to the remaining two groups ol animals on day
  • cecal tissue specimens were removed liom animals which died in the P ⁇ e-24 and P ⁇ e-48 groups I ollowmg the completion ol the study, a lepiesentativeonvmg animal was sacrificed and cecal tissue specimens were removed from the CTAB-24 and CTAB-48 groups. A single untreated animal from the same shipment as those used in the study was also sacrificed and a cecal tissue specimen was removed as a normal control. All tissue specimens were fixed overnight at 4°C in 10% buffered formalin. The fixed tissues were paraffin-embedded, sectioned, and mounted on glass microscope slides. T he tissue sections were then stained using hematoxylin and eosin (H and E stain), and were examined by light microscopy.
  • H and E stain hematoxylin and eosin
  • fhe toxin A gene has been cloned and sequenced. and shown to encode a protein of predicted MW of .308 kd. [Dove et al . Infect. Immun... 58:480-488 ( 1990).
  • the isolated recombinant protein would be soluble in order to preserve native antigenicity. since solubihzed inclusion body proteins often do not fold into native conformations.
  • the recombinant protein should be expressed to levels greater than 1 mg/liter of E. coli culture.
  • toxin A protein can be produced in E. coli.
  • fragments of the toxin A gene were cloned into various prokaryotic expression v ectors, and assessed for the ability to express recombinant toxin A protein in E. coli.
  • Three prokaryotic expression systems were utilized. These systems were chosen because they drive expression of either fusion (pMALc and pGEX2T) or native ( pET23a-c ) protein to high levels in E. coli. and allow affinity purification of the expressed protein on a ligand containing column. Fusion proteins expressed from pGEX vectors bind glutathione agarose beads, and are eluted with reduced glutathione.
  • pMAL fusion proteins bind amylose resin, and are eluted with maltose.
  • a poly-histidine tag is present at either the N-terminal (pETT 6b) or C-terminal (pET23a-c ) end of pET f usion proteins. This sequence specifically binds NL chelate columns, and is eluted w ith imidazoie salts. Extensive descriptions of these vectors are available [ Williams ei al. ( 1995) DNA Cloning 2: Expression Systems. Glover and Hames. eds. I L Press. Oxford, pp. 1 5-58], and will not be discussed in detail here.
  • the Example involved (a) cloning of the toxin A gene, ( b) expression of large fragments of toxin A in various prokaryotic expression systems, (c) identification of smaller toxin A gene fragments that express efficiently in /;. coli. (d) purification of recombinant toxin A protein by affinity chromatography. and (e) demonstration of functional activity of a recombinant fragment of the toxin A gene.
  • a restriction map of the toxin A gene is shown in Figure 6.
  • fhe encoded protein contains a carboxy terminal ligand binding region, containing multiple repeats of a carbohydrate binding domain, ( von Eichel-Streiber and Sauerborn. Gene 96: 107- 1 1 3 ( 1990).]
  • the toxin A gene was cloned in three pieces, by using either the polymerase chain reaction (PCR) to amplify specific regions, ( regions 1 and 2. f igure 6) or by screening a constructed genomic library for a specific toxin A gene fragment ( region 3. f igure 6).
  • prokaryotic expression vectors that express either fusion (pMALc and pGEX2T) or native (pET23a-c) protein to high levels in E. coli. and allow affinity purification of the expressed protein on a ligand containing column.
  • Clostridium difficile VPI strain 10463 was obtained from the AT CC ( ATCC #43255 ) and grown under anaerobic conditions in brain-heart infusion medium ( BBL). High molecular-weight C difficile DNA was isolated essentially as described by Wren and Tabaqchali ( 1987) .1. Clin. Microbiol.. 25:2402. except proteinase K and sodium dodecyl sulfate (SDS) was used to disrupt the bacteria, and cetyltrimethylammonium bromide precipitation [as described in Ausubel et al.. Current Protocols in Molecular Biology ( 1989)] was used to remove carbohydrates from the cleared lysate.
  • SDS sodium dodecyl sulfate
  • genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel. fragments 1 and 2 were cloned by PCR. utilizing a proofreading thermostable DNA polymerase ( native /?/// polymerase; Stratagene). The high fidelity of this polymerase reduces the mutation problems associated with amplification by error prone polymerases (e.g. , Tac j polymerase). PCR amplification was performed using the indicated PCR primers ( Figure 6) in 50 ⁇ l reactions containing 10 mM Tris-HCI(8.3), 50 mM KC1. 1 .5 mM MgCE.
  • Both clones are predicted to produce in-frame fusions with either the glutathione-S-transfcrase protein (pGFX v ector) or the maltose binding protein ( pMAL vector).
  • Recombinant clones were isolated, and confirmed by restriction digestion, using standard recombinant molecular biology techniques. I Sambrook et al.. Molecular Cloning. A Laboratory Manual ( 1989). and designated pGA30- 660 and pM ⁇ 660- l 100. respectiv ely (see figure 6 for description of the clone designations).
  • f ragment 3 was cloned from a genomic library of size selected PstI digested
  • Piotein expression from the three expression constructs made in (a) was induced, and analyzed by Western blot analysis with an affinity purified, goat polycional antiserum directed against the toxm A toxoid ( lech Lab). The procedures utilized for protein induction. SDS-
  • lanes 1-3 contain cell lysates piepared from / coli strains containing pPAl 100-2860 in B!21(DL3)lysE cells: lanes 4-6 contain cell lysates piepared from L coli stiains containing pPAl 100-2860 in B12l(DL3)ly sS cells, lanes 7-9 contain cell h sales prepared from h eo strains containing pMA30-660.
  • lanes 10-12 contain cell lysates prepared liom L coli strains containing pMA660-l 100 fhe lanes were probed with an affinity purified goat antitoxin A polycional antibody (lech Lab). Control lysates from uninduccd cells (lanes 1.7. and 10) contain very little immunoreactive material compared to the induced samples in the remaining lanes. The highest molecular weight band observed for each clone is consistent with the predicted size of the full length fusion protein.
  • Each construct directs expression of high molecular weight HMW) protein that is reactive with the toxin A antibody.
  • the size of the largest immunoreactive bands from each sample is consistent with predictions of the estimated MW of the intact fusion proteins. This demonstrates that the three fusions are in-frame, and that none of the clones contain cloning artifacts that disrupt the integrity of the encoded fusion protein.
  • the Western blot demonstrates that fusion protein from the two larger constructs ( pGA30-660 and pPA l 1 00- 2680) are highly degraded. Also, expression levels of toxin A proteins from these two constructs are low. since induced protein bands are not visible by Coomassie staining (not shown ).
  • interval refers to any portion (i. e.. any segment of the toxin which is less than the whole loxin molecule) of a clostridial toxin.
  • interval refers to portions of C difficile toxins such as toxin A or toxin B. It is also contemplated that these intervals will correspond to epitopes of immunologic importance, uch as antigens or immunogens against which a neutralizing antibody response is effected. It is not intended that the present invention be limited to the particular intervals or sequences described in these Examples. It is also contemplated that sub-portions of intervals
  • compositions and in the methods of the present invention e.g., an epitope contained within one interval or which bridges multiple intervals.
  • Lanes 3 and 4 contain MBP fusion protein purified by solubihzation of msoluble inclusion bodies
  • the purified fusion protein samples are pMA 1870-2680 ( lane 1 ).
  • pM ⁇ 660- l 100 (lane 2 ).
  • pMA30 ⁇ -600 ( lane s ) and pM ⁇ 1450- 1870 (lane 4)
  • T he carboxy terminal end consisting of the repeating units contains the hemagglutination activity or binding domain of C difficile toxin A.
  • hemagglutination assays were performed. Two toxin A recombinant proteins, one containing the binding domain as either soluble affinity purified protein (pMA 1870-2680) or SDS solubihzed inclusion body protein ( pPA l 870-2680) and soluble protein from one region outside that domain (pMA I 100- 1610) were tested using a described procedure. [H.C. rivan et. al . Infect. Immun.. 53:573 ( 1986). I t itrated rabbit red blood cells ( RRBO(Cocalico) were washed several times with
  • Tris-bu fer 0. 1 M Tris and 50 mM NaCl ) by centrifugation at 450 x g for 10 minutes at 4° C.
  • a 1 % RRBC suspension was made from the packed cells and resuspended in T ris-buffer. Dilutions of the recombinant proteins and native toxin A ( T ech Labs) were made in the T ris- buffer and added in duplicate to a round-bottomed 96-well microtiter plate in a final volume of 1 00 ⁇ l. To each w ell. 50 ⁇ l of the 1 % RRBC suspension was added, mixed by gentle tapping, and incubated at 4°C for 3-4 hours.
  • T he expression o ⁇ recombinant toxin A protein as multiple fragments in E.coli has demonstrated the feasibility of generating toxin A antigen through use of recombinant methodologies ( E.xample 1 1 ).
  • the isolation of these recombinant proteins allows the immunoreactivity of each individual subregion of the toxin A protein to be determined ( i. e.. in a antibody pool directed against the native toxin A protein).
  • T his identifies the regions ( if any) for which little or no antibody response is elicited when the w hole protein is u.sed as a immunogen.
  • Antibodies directed against specific fragments of the toxin A protein can be purified bv affmitv chromatography against recombinant toxin A protein, and tested foi neutralization ability This identifies anv toxm A subregions that are essential foi producing neutralizing antibodies Comparison with the levels of immune response directed against these intervals when native toxin is used as an immunogen predicts whether potentially higher 5 liters ot neutralizing antibodies can be produced by using recombinant protein directed against a individual tegion lather than the entire protein I inallv since it is unknown whether antibodies leuctive to the recombinant toxin A proteins produced in 1 xample 11 neutiahze toxin A as effectively as antibodies laised against native toxm ⁇ (Examples 9 and 10) the protective ability oi a pool ot antibodies affinity purified against recombinant toxin A
  • This Example involved (a) epitope mapping of the toxm A protem lo determine the titie ol specific antibodies directed against individual subregions ot the toxin A protein when native toxm v piotein is used as an immunogen. (b) affinity purification of IgY leactive against iecombinant proteins spanning the toxin A gene (e) loxin A neutralization assays with
  • I ⁇ alllnitv potentiation ot whether complete neutralization of toxin A can be elicited with a mixtuie ol antibodies icaclive to iecombinant toxin A protein
  • Ihe iecombinant proteins coiresponding to these intervals were liom expiession clones (see 1 xample I 1(d) foi clone designations) pMA30-3()0 (interval 1) pMA300-660 (interval 2). pMA660-l 100 (interval 3). pPAl 100-1450 (interval 4). pMA1450-1870 (interval 5) and pMA 1870-2680 (interval 6) These 6 clones were selected because they span the entire protein trom amino acids numbered 30 through 2680. and subdivide the protein into 6 small intervals Also, the carbohydrate binding repeat interval is contained specifically in one interval (interval 6).
  • Bound antibody was eluted from the column by w ashing with 5 column volumes of 4 M Guanidine-HCl (in 10 mM T ' ris- I ICI. pl l 8.0). T he column was reequilibrated in PBS. and the depleted antibody stock reapplied as described above, The eluate was collected, pooled w ith a 1 ml PBS wash, quantitated by absorbance at D-> S(1 . and stored at 4° ( ' . In this manner. 6 aliquots of the CTA IgY preparation were individually depicted for each of the 6 toxin A intervals, by two rounds of affinity depletion. The specificity of each depleted stock was tested by Western blot analysis.
  • Region specific antibody pools were isolated by affinity chromatography as described below, Pen mis of the dialyzed CTA IgY preparation were applied sequentially to each affinity column, such that a single 10 ml aliquot was used to isolate region specific antibodies specific to each of the six subregions. The columns were sequentially washed with 1 0 volumes of PBS. 6 volumes of BBS-Tween. 10 volumes of TBS. and eluted with 4 l ⁇ ctisep elution media (Sterogene).
  • T he eluate was dialyzed extensively against several changes of PBS. and the affinity purified antibody collected and stored at 4°C.
  • the volumes of the eluate increased to greater than 10 mis during dialysis in each case, due to the high viscosity of the Actisep elution media.
  • Aliquots of each sample were 20x concentrated using Centricon 30 microconcentrators (Amicon) and stored at 4°C.
  • the specificity of each region specific antibody pool was tested, relative to the dialyzed CTA IgY preparation, by Western blot analysis, exactly as described above, except that 4 ml samples of blocking buffer containing 100 ⁇ l region specific antibody (unconcentrated ) were used instead of the depleted CTA IgY preparations.
  • Each affinity purified antibody preparation was specific to the defined interval, except that samples purified against intervals 1 -5 also reacted with interval 6. This may be due to non-specific binding to the interval 6 protein, since this protein contains the repetitiv e ligand binding domain which has been show n to bind antibodies nonspeciflcallv. I Lyerly et al . Curr. Microbiol.. 19:303-306 ( 1989).
  • fhe reactivity of each affinity purified antibody preparation to the corresponding proteins was approximately the same as the reactivity of the 1/500 diluted dialyzed CTA IgY preparation standard. Given that the specific antibody stocks were diluted 1 /40. this would indicate that the unconcentrated affinity purified antibody stocks contain 1 / 1 0- 1 /20 the concentration of specific antibodies relative to the starting CT A IgY preparation.
  • T he soluble and the inclusion body preparations each were used separately to immunize hens. Both purified toxin A polypeptides were diluted in PBS and emulsified with approximately equal volumes of CFA for the initial immunization or IFA for subsequent booster immunizations.
  • IgG (Sigma) diluted 1 : 1000 in antibody diluent was added to the plate, and incubated for 1 hour at 37°C. The plates were washed as before and substrate was added, [p-nitrophenyl phosphate ( S ⁇ gma)
  • ELISA titers ( reciprocal of the highest dilution of IgY generating a signal) in the preimmune IgY to the MBP or the recombinant was ⁇ 1 :30 while the immune IgY titers to MBP and the toxin A recombinant were 1 : 1 8750 and > 1 :93750 respectively.
  • the anti-reeombinant antibody titers generated in the hens against the recombinant polypeptide is much higher, compared to antibodies to that region raised using native toxin A.
  • toxin A agglutinates rabbit erythrocytes by binding to a trisaccharide ( gal 1 -3131 - 4GlcNAc) on the cell surface.
  • a trisaccharide gal 1 -3131 - 4GlcNAc
  • W e examined whether the anti-recombinant toxin A ( immune IgY. antibodies raised against the insoluble product expressed in pET) can neutralize the hemagglutination activity of toxm A in vitro, fhe hemagglutination assay procedure used was described by I EC. Krivan el al.
  • Polyethylene glycol-fractionated immune or preimmune IgY were pre-absorbed w ith citrated rabbit erythrocytes prior to performing the hemagglutination assay because we have found that IgY alone can agglutinate red blood cells.
  • RRBC ⁇ s Citrated rabbit red blood cells (RRBC ⁇ s)(Coeaiico) were washed twice by centrifugation at 450 x g with isotomc buffer (01 M T ⁇ s-HCI.005 M NaCl pH 72) RRBC-ieactive antibodies in the IgY were removed bv preparing a 10% RRBC suspension (made bv adding packed cells to immune oi preimmune IgY) and incubating the mixture toi 1 houi at 37°C The RRBCs weie then removed bv centrifugation s Neutiahzation of the hemagglutination activity of toxin A bv antibodv was tested in lound- bottomed 96-well mictotiter plates Iwentv-flve ⁇ l ot toxm A (36 ⁇ g /ml) (Tech Lab) in isoto c butter was mixed with an equal volume of different dilutions ol immune or prei
  • Ihe abihtv ol the anli-recombinant toxin A IgY (immune IgY antibodies uused against 0 p ⁇ l ⁇ I870-2680 the soluble iecombinant binding domain piotein expressed in p ⁇ l ⁇ L designated as Anti-tox ⁇ -2 in 1 iguie 14 and referred to as iecombinant icgion 6) and pie- immune ⁇ piepaied as desc ⁇ bed in E ample 8(c) above to neutralize the cvtotoxic activity ol toxin using the CHO cell cvtotoxicitv assav and toxm A ( lech 1 ub) at a eoncentiation ol 0 lug ml as described m
  • Example 8(d) above ⁇ s additional s controls the anti-native toxm ⁇ Ig (CTA) and pre-immune IgY prepaiations described in
  • the ability oi avian antibodies (IgY) raised against iecombinant toxm A binding domain to neuttahze the enterotoxin activity oi ( difficile toxin A was evaluated in vivo using Golden Svnan hamsters
  • the Example involved (a) preparation of the avian anti- leeombinant toxin A IgY for otal administration (b) in ⁇ o protection of hamsteis trom ( difficile toxm A entcrotoxicitv bv treatment ot toxm A with avian anti-reeombinant toxm A Is_Y and (e) histolomc evaluation of hamster ceca
  • the second group consisted ot two animals which were not part of the studv described above but were separately treated with the same toxin A t pre-immune IgY mixtures as desciibed lor the animals in section (b) above Both ot these hamsters developed diarrhea, and were sacrificed 8 hrs after the time of admmistiation ot the toxin A - pie-immune IgY mixtuies ⁇ t the time ol sacrifice both animals were presenting symptoms ol diarrhea These animals icpresented the acute phase ol the studv
  • Ihe ⁇ ma] group consisted ot a single untreated hamster tiom the same shipment ol animals as those used lor the two previous groups I his animal served as the normal control
  • Intestinal vilh and crypts were significantly diminished and appealed to have been overgrown bv a planai laver ot epithelial cells and llbroblasts Iheretoie although these animals ovcrtlv appealed to lecover tiom the acute symptoms ot toxm A intoxication lasting pathologic alterations to the cecal mucosa had occurred
  • Ihe tissues obtained liom the two acute animals which received mixtuies of tox A and pie-immune IgY demonstrated the most significant pathology ⁇ l the gross pathological level both animals eie observed to have severely distended ceca which were filled with watery diairhea-hke material ⁇ t the microscopic level, the animal that was given the mixture containing l O ⁇ g of toxin A and pre-immune IgY was found to have a mucosal layer w hich had a ragged, damaged appearance, and a disorganized, compacted quality.
  • the crypts were largely absent, and numerous breaks in the epithelium had occurred. There was also an influx of erythrocytes into spaces between the epithelial layer and the underlying tissue.
  • the animal w hich had received the mixture containing 30 ⁇ g of toxin A and pre-immune IgY demonstrated the most severe pathology.
  • the cecal tissue of this animal had an appearance v ery similar to that observed in animals which had died from C. difficile disease. Widespread destruction of the mucosa was noted, and the epithelial layer had sloughed. I Iemorrhagic areas containing large numbers of erythrocytes were very prevalent. All semblance of normal tissue architecture was absent from this specimen.
  • T he ability of avian antibodies directed against the recombinant ( ' . difficile toxin A fragment 1 70-2680 (as expressed by pMA 1870-2680: see E.xample 13 ) to neutralize the enteroto.xic activity of toxin A was demonstrated in E.xample 14.
  • T he ability of avian antibodies ( IgYs) directed against other recombinant toxin A epitopes to neutralize native to in A in vivo was next ev coed.
  • This example involved: (a) the preparation of " IgYs against recombinant toxin A poly peptides: (b) in vivo protection of hamsters against toxin A by treatment with anti-reeombinant toxin A IgYs and (c) quantification of specific antibody concentration in CTA and Interval 6 IgY PEG preparations.
  • T he nucleotide sequence of the coding region of the entire toxin A protein is listed in SEQ I D NO:5.
  • the amino acid sequence of the entire toxin A protein is listed in SEQ II) O:6.
  • T he amino acid sequence consisting of amino acid residues 1 870 through 2680 of toxin A is listed in SEQ ID NO 7
  • the amino acid sequence consisting of am o acid lesidues 1870 thiough 1960 ot toxin A is listed in SFQ ID NO 8
  • I ggs were collected trom 1 eghorn hens which have been immunized with recombinant ( difficile loxin ⁇ polvpcptide tiagments encompassing the entire toxm
  • a protem fhe pol y peptide tiagments used as immunogens were 1) pMA 1870-2680 (Inteival 6) 2) pPA 1100-1450 (Inteival 4) and 3) a mixture of fragments consisting ot p 30-300 (Inteival 1)
  • pM icfers to the pMAI IM -e vector New 1 ngland BioLubs.
  • pG refers to the pGEX vector (Pharmacia):
  • pB refers to the PinPoint IM Xa vector (Promega):
  • A refers to toxin A; the numbers refer to the amino acid interval expressed in the clone.
  • Ihe solid black ovals represent the MBP: the hatched ovals represent glutathione S-transfcrase: the hatched circles represent the biotin tag: and ]] ⁇ U ⁇ represents the poly-histidine tag.
  • An affinity column containing recombinant toxin A icpeat protein was made as follows 1 our ml ol " PBS-washed ⁇ ctigel resin (Sterogene) was coupled with 5-10 mg of pP ⁇ 1870-2680 inclusion body protein
  • each dose of the Inteival 6 antibody prep contained approximately 400 ⁇ g of specific antibodv
  • each hamster was predisposed to ( difficile infection by the oial administration of 30 mg ol " CT ⁇ ndamycm-1 IC1 (Sigma) in I ml
  • Ihe hamsters were first predisposed to ( difficile infection with a 3 mg dose ol Chndamycin-HCT (Sigma) administered orally in 1 ml of water Approximately 24 hrs later, the animals were orally challenged with 1 ml of C difficile strain A fCC 43596 m sterile saline containing approximately 200 organisms. One day after infection, the presence of toxin A and B was determined in the feces ot the hamsters using a commercial immunoassay kit (Cytoelone A-B EPA. Cambridge Biotech) to verify establishment of infection I our members of each group were randomly selected and tested. Teces from an unmfected hamster was tested as a negative control. All infected animals tested positive for the presence of toxin accoiding to the manufacturer ' s procedure The initiation of treatment then started approximately 24 hi post-infection
  • This example involved a) expression ol the toxin A icpeats and sublragments ot these icpeats in / coli using a vancty ot expression vectors b) identification ot iecombinant toxm A repeats and sub-regions to which neutiahzmg antibodies bind and c) determination oi the neutiahzation ability ol antibodies laised aua st soluble and insoluble toxin A icpeat immunogen a) Expression Of The Toxin A Repeats And Subfragments Of
  • the Interval 6 immunogen utilized in Examples 15 and 16 was the pMA 1870-2680 piotem. which the toxin A repeats aic expressed as a soluble fusion protein with the MBP
  • polv -I is tagged fpP ⁇ 1870-2680 (II)] or biotin-tagged (pBA 1870-2680) pioteins lesultcd in completely insoluble protem upon induction ol the baetenal host (see 1 iguie I 5B)
  • the host strain BI 21 (Novagen) was used for expression of pBA 1870-2680 and host stum B( 21(Dr 3) (Novagen) was used foi expiession of pPA1870-2680 and pP ⁇ I870- 2680(11) I hese insoluble pioteins accumulated to high levels in inclusion bodies t xpression ol iecombinant plasmids in / eoli host cells giown in 2X ⁇ medium was perfoimed as desciibed
  • Ihe solubility ol expiessed piole ns Irom the pPG1870-2190 and pP ⁇ 1870-2190 expiession constiucts was determined alter induction ol iecombinant protein expiession undei conditions lepoited to enhance piotem solubility [These conditions comprise gio th ol the host at reduced temperature (3()°C) and the utilization ot high (1 mVl 1PIG) oi low (01 mM 1PTG) concentrations ol mducer [Williams el al (1995).
  • Toxin A repeat regions to w hich neutralizing antibodies bind were identi fied bv utilizing recombinant toxin A repeat region proteins expressed as soluble or insoluble proteins to deplete protectiv e antibodies from a polycional pool of antibodies against native ( ' . difficile toxin A. .An in vivo assay was developed to evaluate proteins for the ability to bind neutralizing antibodies. fhe rational for this assay is as follows. Recombinant proteins were first pre-mixed w ith antibodies against native toxin A (CTA antibody : generated in Example 8 ) and allowed lo react. Subsequently. ( ' .
  • difficile toxin A was added at a concentration lethal to hamsters and the mixture was administered to hamsters via I P injection.
  • I f the recombinant protein contains neutralizing epitopes.
  • the CTA antibodies would lose their ability to bind toxin A l esulting in diarrhea and/or death of the hamsters.
  • T he assay was performed as follows. T he lethal dose of toxin A when delivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco) was determined to be 10 to 30 ⁇ g. T he
  • PEG-puriflcd CTA antibodv preparation was diluted to 0.5X concentration (i. e.. the antibodies were diluted at twice the original yolk volume) in 0. 1 M carbonate buffer. pH 9.5. fhe antibodies were diluted in carbonate buffer to protect them from acid degradation in the stomach. T he concentration of 0.5X was u.sed because it was found to be the lowest effective concentration against toxin A. T he concentration of Interval 6-specifie antibodies in the 0.5X
  • CT A prep w as estimated to be 10- 1 5 ⁇ g/ml (estimated using the method described in Example 1 5 ).
  • the inclusion body preparation insoluble Interval 6 protein: pPA l 870-2680(11)] and the soluble Interval 6 protein [pMA 1870-2680: see figure 15] were both compared for their ability to bind to neutralizing antibodies against C. difficile toxin A (CTA).
  • CTA C. difficile toxin A
  • 1 to 2 mg of recombinant protein was mixed with 5 ml of a 0.5X CTA antibody prep (estimated lo contain 60-70 ⁇ g of Interval 6-specillc antibody ). After incubation for 1 hr at 37°C.
  • C difficile lox in A was added to each group.
  • An imals showed no signs ot " i l lness.
  • a nimals developed diarrhea but did not die. Animals developed diarrhea and died.
  • Preimmune antibody was ineffective against toxin A. while anti-C f A antibodies at a dilute 0.5X concentration almost completely protected the hamsters against the enterotoxic effects ol " CTA.
  • the addition of recombinant proteins pPB 1 850-2070 or pPA 1 870-2 1 0 to the anti-CTA antibody had no effect upon its protective ability, indicating that these recombinant proteins do not bind to neutralizing antibodies.
  • Ihe sonicated suspension was tianslerred to a 35 ml Oakndge tube and eeni ⁇ li ed at 6000 X g lor 10 mm at 4°C to pellet the inclusion bodies Ihe pellet was washed 2 times bv pipetting oiroisexing in iiesh.
  • the samples were diluted with 9 volumes ot PBS Ihe protem solutions were dialvzed overnight against a 100-fold volume ol PBS containing 005% SDS at loom temperature
  • the dialvsis buffer was then changed to PBS containing 001% SDS and the samples were dialyzed for several hours to overnight at room tempeiature
  • the samples were stored at 4°C until used P ⁇ oi to further use.
  • the samples were w imed to loom temperature to allow anv piecipitatcd SDS to go back into solution
  • Ihe inclusion body preparation was used to immunize hens fhe protem was diaivzed into PBS and emulsified with approximately equal volumes ot CTA lor the initial immunization oi If ⁇ lot subsequent booster immunizations On dav zeio lor each of the iecombinant iecombinant preparations, two egg laving white I eghorn hens were each injected at multiple sites (IM and SC) with 1 ml of recombinant proiei ⁇ -ud ⁇ ivant mixtuie containing approximatelv 05-1 5 mg ol recombinant protein Booster immunizations of 10 mg were given ol davs 14 and dav 28 I ggs were collected on dav 32 and the antibodv isolated using PI G as desciibed in I xample 14(a) High titers ol toxin ⁇ specific antibodies were present (as assayed bv 1 I 1
  • TABLF 22 cuti.ili/.ition Ol loxin A Bv Antibodies Auainst Soluble Interval 6 Piotein Studv Outcome ⁇ ltci 24 Houis
  • Antibodies raised against native loxin A were protective while preimmune antibodies had little etlect As found using the in CHO assay [described in f xample 8(d)) where antibodies laised against the soluble Interval 6 could paitiallv neutiahze the etfects ol toxin A here thev were able to completely neutiahze toxin A in In contrast, the antibodies raised against the insoluble Interval 6 was unable to neutralize the effects of toxin A in vivo as shown above (Table 22) and in vitro as shown in the CHO assay [described in Example 8(d)].
  • the amino acid sequence ol the eniite toxin B protein is l isted in SEQ ID NOT 0 I he ammo acid sequence consisting ol ammo acid residues 1 850 through 2360 ot toxin B is listed in SEQ I D NO 1 1 I he ammo acid sequence consisting ol amino acid residues 1 750 through 2360 ot toxin B is l isted in SI Q ID NO- 12 Giv en the expense and difficulty of isolating native toxin B protein, il would be adv antageous lo use simple and inexpensiv e procaryotic expression sy stems lo produce and purify high lev els ol recombinant toxin B protein lor immunization purposes Ideally , the isolated recombinant protem would be soluble in ol der to preserv e nativ e anligenicity .
  • ince solubihzed inclusion bodv proteins often do not fold into nativ e eonlormations Indeed as show n in Example 1 7.
  • neutralizing antibodies against recombinant toxin A were e only obtained w hen soluble recombinant toxin A polypeptides were used as the immunogen.
  • l o allow ease ol puri fication, the recombinant protein should be expressed to lev els g ⁇ eater than 1 mg/hter of E coli culture. fo determine w hether high lev els of recombinant toxin B protein could be produced in I. c oh.
  • ti agments of the toxin B gene were cloned into various prokary otic expression v ectors, and assessed for the ability to express recombinant toxin B protein in E coli I his f xample inv olv ed (a) cloning of the toxin B gene and (b) expression of the toxin B gene in /: co . a) Cloning Of The Toxin B Gene
  • the toxin B gene was cloned using PCR amplification from C. difficile genomic DNA. Initially, the gene was cloned in two overlapping fragments, using primer pairs P5/P6 and P7/P8. The location of " these primers along the toxin B gene is shown schematically in figure 18. The sequence of each of these primers is: P5: 5 " TAGAAAAAATGGC ⁇ TGT 3 " (SEQ ID NO: 5 " TAGAAAAAAAATGGC ⁇ TGT 3 " (SEQ
  • figure 18 also shows the location of the following primers along the loxin B gene: P9 which consists of the sequence 5 ' CGGAATTCCTAGAAAAAATGGCA ⁇ ATG 3 " (SEQ ID NO: 12): P7: 5 " GATGCC ⁇ CA ⁇ GATGATTT ⁇ GTG 3 * (SEQ ID NO: I 3): and P8: 5 " CTAATTGAGCTGTATCAGGATC 3 ' (SEQ ID NO: 14).
  • figure 18 also shows the location of the following primers along the loxin B gene: P9 which consists of the sequence 5 ' CGGAATTCCTAGAAAAAATGGCA ⁇ ATG 3 " (SEQ ID NO: 12): P7: 5 " GATGCC ⁇ CA ⁇ GATGATTT ⁇ GTG 3 * (SEQ ID NO: I 3): and P8: 5 " CTAATTGAGCTGTATCAGGATC 3 ' (SEQ ID NO: 14).
  • figure 18 also shows the location of the following primers along the loxin B gene: P9 which
  • amino acid sequence consisting of amino acid residues 1852 through 2362 of toxin B is listed in SEQ ID NO:20.
  • amino acid sequence consisting of amino acid residues 1755 through 2362 of toxin B is listed in SEQ ID NO:21.
  • Clostridium difficile VPI strain 10463 was obtained from the American Type Culture Collection (ATCC 43255) and grown under anaerobic conditions in brain-heart infusion medium (Becton Dickinson). High molecuiar-weight ( ' . difficile DNA was isolated essentially as described [Wren and Tabaqchaii (1987) J. Clin. Microbiol..25:2402). except I) 100 ⁇ g/ml proteinase k in 0.5% SDS was used to disrupt the bacteria and 2) cetytrimethvTammonium bromide (CTAB) precipitation jas described by Ausubel et al. Eds..
  • CTAB cetytrimethvTammonium bromide
  • the aqueous supernatant was removed and extracted with phenol/chloroform/ isoamyl alcohol (25:24:1 ). The phases were separated by centrifugation in a microfuge for 5 min. The supernatant was transferred to a fiesh tube and the DNA was precipitated with isopropanol The DNA precipitate was pelleted bv bnef centrifugation in a microfuge The DNA pellet was washed with 70% ethanol to remove residual CI ⁇ B Ihe DNA pellet was then dried and ledissolved in TI buffei (10 mM Ins-HCl pH80 I mM I DT ⁇ ) Ihe integiitv and yield ol genomic DNA was assessed bv comparison with a serial dilution ot uncut lambda
  • Piocedures lor protem induction SDS-PAGE and Western blot analysis were performed as described m Williams a al (1995) supia In bnef " ml cultuies ol bacteria giown in 2XYT containing 100 ⁇ g/ml ampiciilin containing the uppiop ⁇ ute iecombinant clone were induced to express recombinant protein by addition of IPTG to ImM.
  • the E. coh hosts u.sed were: BE2KDE3) or BL21(DE3)LysS (Novagen) for pET plasmids.
  • poly-histidine sequence binds to Ni-Chelate columns and allows affinity purification of tagged target proteins [Williams el al (1995), supra].
  • I hese affinity tags arc small (10 aa for pET16b.6 aa for pET23) allowing the expression and affinity purification ol native proteins with only limited amounts ol foreign sequences
  • N-terminal lustidine-tagged derivative of pE I 16b containing an extensive cloning cassette was constructed to facilitate cloning of N-terminal poly-histidine tagged toxin B expressing constructs This was accomplished by replacement of the promoter region oi the pl ' T23u and b vectors with that of " the pET16b expression vector Each vector was restricted with BglU and h'de ⁇ . and the reactions resolved on a 1.2 % agarose gel The pET 16b promoter region (contained in a 200 bp BglU-Nde] fragment) and the promoter-less pET23 a or b vectors were cut trom the gel.
  • MBP lusion proteins were constructed and expressed in the pMAI IM -c or pMAI I -p2 vectors (New England Biolabs) in which the protem of interest is expressed as a C-iermmal lusion with MBP All pET plasmids were expressed in either the BI 21(DE3) or
  • pETT 6b or pETHisa-b expression vectors or by engineering restriction sites at specific locations using PCR amplification [using the same conditions described in (a) above). In all cases, clones were verified by restriction mapping, and. w here indicated. DNA sequencing.
  • Protein preparations from induced cultures of each of these constructs were analyzed, by SDS-PAGE. to estimate protein stability (Coomassie Blue staining) and immunoreactivity against anti-toxin B specific antiserum ( Western analysis). Higher levels of intact ( i. e. . nondegraded). full length fusion proteins were observed with the smaller constructs as compared with the larger recombinants. and a series of expression constructs spanning the entire toxin B gene were constructed ( figures 1 8. 19 and 20 and T able 23).
  • T hese clones were utilized to isolate pure toxin B recombinant protein from the entire to m B gene.
  • Significant protein y ields w ere obtained from pMAE expression constructs spanning the entire toxin B gene, and yields o ⁇ full length soluble fusion protein ranged from an estimated I mg/liter culture (pMB l 100- 1530) to greater than 20 mg/liter culture (pMB 1750-2360).
  • Representative purifications of MBP and poiy-histidine-tagged toxin B recombinants are show n in figures 21 and 22.
  • f igure 21 shows a Coomassie Blue stained 7.5% SDS- PAGE gel on which v arious protein samples extracted from bacteria harboring pMB I 50- 2360 were electrophoresed. Samples were loaded as follows: Eane 1 : protein extracted from uninduced culture: Eane 2: induced culture protein: Eane 3 : total protein from induced culture after sonication: Lane 4: soluble protein: and Lane 5: eluted affinity purified protein, figure
  • gel purified pMALc or pFT23b vector I hese v ectors were prepared by digestion w ith IlindWl. filling in the over hanging ends using the Klenow enzyme, and cleaving with Xbal ( pMALc) or Nhel ( pET23b)
  • the gel purified DNA liagments were purified using the Prep-A-Gene kit ( BioRad ) and the DNA was ligated.
  • transtoi med and putative recombinant clones analvzed by restriction mapping pi 1 and pMal clones containing the toxin B lepeat inser t were verified by restriction mapping, using enzvmes that cleaved specific sites within the toxin B icgion
  • fusion ot the toxin B .S/;el site w ith either the compatible Yha site (pMal) or compatible ⁇ hel site (pET) is predicted to create an in frame tusion I his was confirmed in the case of the pMB l 750-2360 clone by DNA sequencing ot the clone junction and 5 ' end of the toxin B insert using a MBP specific primer (New England Biolabs).
  • the fusion of the blunt ended toxin B 3 " end to the filled Hindlll site should create an in-frame fusion with the C-terminal poly-histidine sequence in this vector.
  • the pPB 1750-2360 clone selected had lost, as predicted, the Hindlll site at this clone junction: this eliminated the possibility that an additional adenosine residue was added to the 3 ' end of the PCR product by a terminal transferase activity of the Pfu polymerase. since fusion of this adenosine residue to the filled Hindlll site would regenerate the restriction site (and was observed in several clones).
  • affinity purification resulted in yields in excess of 20 mg protein per liter culture, of which greater than 90% was estimated to be full-length recombinant protein.
  • the poly-histidine affinity tagged protein was released from the Qiagen Ni-NTA resin at low imidazoie concentration (60 mM ). necessitating the use ol " a 40 mM imidazoie rather than a 60 mM imidazoie wash step during purification.
  • a periplasmically secreted v ersion of pMB l 750-2360 was constructed by replacement of the promoter and MBP coding region of this construct with that from a related vector ( pMAI. I N '-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP. such that fusion protein is exported. This was accomplished by substituting a related vector ( pMAI. I N '-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP. such that fusion protein is exported. This was accomplished by substituting a related vector (pMAI. I N '-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP. such that fusion protein is exported. This was accomplished by substituting a related vector (pMAI. I N '-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP
  • Bgll -EcoRM promoter fragment from pMAL-p2 into pMB l 750-2360 The yields of secreted, affinity purified protein ( recovered from osmotic shock extracts as described by Riggs in Current Protocols in Molecular Biology. Vol. 2. Ausubel. el al.. Eds. ( 1989). Current Protocols, pp. 16.6. 1 - 16.6. 14] from this vector (pMBp! 750-2360) were 6.5 mg/liter culture. of which 50% was estimated to be full-length lusion protein. fhe interval was also expressed in two non-overlapping fragments.
  • pMB 1 750- 1970 was constructed by introduction of a frameshift into pMB l 750-2360.
  • the complementary region was expressed in pMB 1970-2360 fins construct was created bv icmoval ot the 1750-1970 interval of pMB I 750-2360 I his was accomplished by lestrietion ol this plasmid with EcoRl (in the pMalc polylinker 5 to the insert) and III. filling in the overhanging ends, and rehgation ol the plasmid The resultant plasmid.
  • pMB 1970-2360. was made using both intracellularly and secreted versions ol the pMBl 750-2360 vector No fusion protein was secreted in the pMBp 1970-2360 veision. perhaps due to a contoimational constraint that prevents export of the fusion protem However, the intracellularly expressed vector produced greater than 40n ⁇ g/hter of greater than 90% full- length lusion protein
  • the amplified Iragment was cloned into the pi 7 Blue I -vector (Novagen) and recombinant clones in which single copies ot the PCR tiagment were inserted in either onentation were selected (p I 71850-2360) and confirmed by restiiction mapping Ihe insert was excised tiom two appropriately oriented independently isolated pi 71850-2360 plasmids.
  • the lesultant construct (pMBl 850- 1970) expressed greater than 70 mg/hter ot 90% lull length fusion piotem
  • the pPB 1 850-2360 construct was made by cloning a EcoRl (filled with Klenow)- Ba l fragment from a pT71850-2360 vector (opposite orientation to that used in the pMB 1850-2360 construction) into Ndel (filledVZ ⁇ wHI cleaved pETl ⁇ b vector. Yields of " affinity purified soluble fusion protein were 15 mg/liter. of greater than 90% full length fusion protein.
  • pPB l 850-2070 was constructed by cloning a BglU-Pvull fragment of pPB l 850-2360 into Bglll/Hincl l cleaved pET23b vector.
  • pPB 1 750- 1970(c ) was constructed by removal of the internal Hindlll fragment of a pPB 1 750-2360 v ector in which the vector Hindlll site was regenerated during cloning (presumably by the addition of an A residue to the amplified PCR product by terminal transferase activity o ⁇ Pfu polvmerase).
  • An expression construct that directs expression of the 10-470 aa interval of toxin B was constructed in the pMalc vector as follows.
  • a Nhe (a site 5 " to the insert in the pET23 vector )-A fl] 1 (filled) fragment of " the toxin B gene from pPB 10- 1530 was cloned into Xbal ( compatible w ith ⁇ ' hel )l Hindlll (filled) pMalc vector.
  • T he integrity of the construct ( pMB I O- 470) was verified by restriction mapping and DNA sequencing of the 5 ' clone junction using a MBP specific DNA primer ( Ne England Biolabs). However, all expressed protein was degraded to the MBP monomer MW.
  • a second construct spanning this interval (aa 10-470) was constructed by cloning the PCR amplification product from a reaction containing the P9 (SEQ ID NO: I 5 ) and P 10 (SEQ ID NO: 16) primers ( Figure 18) into the pETHisa vector. This was accomplished by cloning the PCR product as an /: ' cv;RI-blunt fragment into EcoRl-Hinc ⁇ l restricted vector DNA: recombinant clones were verified by restriction mapping. Although this construct (pPB I O-
  • the pMB260-520 clone was constructed by cloning EcoRl- Xbal cleaved amplified DNA tiom a PCR reaction containing the PI 1 (SfQ ID NO 17) and Pl ⁇ (SEQ ID NO 16) DNA pnmers (1 igure 18) into similarly lest ⁇ ctcd pMalc vector Yields ol affinity purified protem were 10 mg/hter ot which approximately 50% was estimated to be lull-length iecombinant protein
  • Ihe aa5I0-l 110 interval was expressed as described below 1 his entne interval was expressed as a pMal lusion bv cloning the hhel-Hmdlll fragment ot pUCBlO-1530 into Xhal- I ndlll cleaved pMalc vector
  • Ihe integrity of the construct (pMB510-l 110) was verified by lestrietion mapping and DNA sequencing of the 5 clone junction using a MBP specific DNA primer
  • Ihe scord ol affinity purified protem was 25 mg/hter cultuie ol which 5% was estimated to be lull-length lusion protein (1 mg/hte ) lo attempt to obtain higher yields, this region was expressed two tiagments (aa510- 820 and 820-1110) in the pMalc vector 1 he pMB510-820 clone was constiucted by inset tion ol a Sad (in
  • the aal 100-1750 interval was expressed as described below The entire interval was expressed in the pMalc vector from a construct in which the tT(fiIled)- /?t'I fragment of pPB10-1750 was inserted into Slul/Xhal (Xbal is compatible with Spel.
  • I hree constructs were made to express the remaining interval Initially, a BspHl (fllied)-.S/;el fragment fiom pPBlO-1750 was cloned into £ cleaved pMalc veetoi Ihe integrity ol this construct (pMB1570-l 750) was verified by restriction mapping and DNA sequencing of the 5 " clone junction using a MBP specific DNA primer 1 xpression ol iecombinant protein from this plasmid was very low.
  • Antibodies against CTB were generated in egg laying Leghorn hens as described in Example 8
  • the lethal dosage ( LD , prescribil) of C difficile toxin B when delivered I.P into 40g female (Jolden Syrian hamsters (Charles River) was determined to be 2.5 to 5 ⁇ g.
  • Antibodies generated against CTB and purified by PEG precipitation could completely protect the hamsters at the I P dosage determined above
  • the minimal amount of CTB antibody needed to af ford good protection against 5 ⁇ g of CTB when injected I P into hamsters was also determined ( I X PEG prep).
  • vv hethei recombinant toxin B poly peptide fragments can generate neutralizing antibodies.
  • Iv picalh animals would first be immunized with recombinant proteins and anti-i ecombinant antibodies ate generated 1 hese anti-recombmant protein antibodies are then tested toi neuti ahzmg ability in vivo or in vin o
  • anti-i ecombinant antibodies ate generated 1 hese anti-recombmant protein antibodies are then tested toi neuti ahzmg ability in vivo or in vin o
  • the i ecombinant polypeptide the generation of high-titer antibodies against that protein may take several months l accelerate this process and identify which recombinant polypeptide!
  • s may be the best candidate to generate neutralizing antibodies, depletion studies ei e pei f ormed Specifically , i ecombinant toxin B polypeptide were pre-scieened bv testing vv hethei thev hav e the ability to bind to protective antibodies trom a CTB antibodv prepai ation eind hence deplete those antibodies of their neutralizing capacity I hose recombinant polv peptides found to bind CT B, were then utilized to generate neutralizing antibodies 1 his Example involved a) identification of recombinant sub-iegions within toxin B to w hich neutralizing antibodies bind, b) identification ot toxin B sub-region specific antibodies that neutralize toxin B in vivo, and c) generation and ev aluation ot antibodies icaetiv e to i ecombinant toxin B poly peptides
  • pPB 1750-2360 and pPB 1850-2360 can bind and completely remove neutralizing antibodies from the CTB antibody pool.
  • Recombinant proteins comprising only a portion of the toxin B repeat domain were not capable of completely removing neutralizing antibodies from the CTB antibody pool (pMB l 750- 1970 and pMB l 970-2360 could partially remove neutralizing antibodies while pMB 1850- 1970 and pPB 1850-2070 failed to remove any neutralizing antibodies from the C TB antibody pool ).
  • T hese results represent the first indication that the entire repeat region of toxin B would be necessary for the generation of antibodies capable of neutralizing toxin B. and that sub-regions may not be sufficient to generate maximal titers of neutralizing antibodies.
  • T he eluted antibody was 0 immediatel y dialyzed against a 100-fold excess of PBS at 4°C for 2 hrs fhe samples were then dialv zed extensively against at least 2 changes ot PBS.
  • affinity purified antibody was collected and stored at 4°C T he antibody preparations wei e quantified by UV absorbance Hie elution v olumes were in the range of 4-8 ml All affinity purified stocks contained sinulai total antibodv concentrations, ranging f iom 0 25-0 35% ol the total protein applied to ⁇ the columns f he ability of the af Unity purified antibodv preparations to neutralize toxin B m vivo was determined using the assay outlined in a) above Affinity purified antibody was diluted 1 I in PBS betore testing The results are shown in I able 26
  • Tlie amount ol specific antibody in each prep is indicated, tlie amount is direciiv detet mmed foi af f inity punfied preps and is estimated foi the 4X C I B as described in l xample 1 >
  • T he numbers in each uroup represent numbers ot hamsters dead oi alive 2 hr post IP administration of toxin/antibodv mixture
  • 96-wcll microtitet plates I aleon. Pro-Bind Assay Plates
  • recombinant piotein by adding 100 ⁇ l volumes oi protein at 1 -2 ⁇ g/ml m PBS containing 0.005% thimerosal to each vveil and incubating ov ernight at 4°C I he next morning, the coating suspensions were decanted and the wells weie washed thiee times using PBS In order to block non-specific binding sites.
  • Figure 24 depicts a comparison of immunoreactivity of IgY antibody raised against either native or recombinant toxin B antigen Equal amounts of pMB1750-1970 (lane 1), pMB 1970-2360 (lane 2). pPB 1850-2360 (lane 3) as well as a serial dilution of pPB 1750-2360
  • Antibodies against recombinant proteins were generated in egg laving Eeghom hens as described in Example 13 Antibodies were raised
  • pMBl 750-2360 (6-6) pMBl 750-2360 [Iite ⁇ nax adiuvant (Vaxcell)].7) pMBl 750-2360 (Gerbu ad ivant (Biotech)] 8) pMBpl750- 2360 piotein: 9) pPBl 850-2360.
  • Ihe numbers m eacli group represent numbers of hamsteis dead oi alive 2 hours post IP admmistiation ot mixture
  • C difficile toxin B (CTB) (at 5 ⁇ g'inl. 25 ⁇ g total. Tech Lab) at lethal concentration to hamsteis is added to antibodv and incubated for one hour at 37°C After incubation.
  • I ml ol this mixture is miected intrape ⁇ toneally (IP) into hamsters horr treatment group received toxin premixed with antibody raised against the indicated protein, as either (I ) 4X antibody PEG prep or (2) affinity purified antibody (on a pPB 1750-2360 resin), either I 5 mg/group (anti-pMB 1750-2360 and anti-p B 1970-2360. used undiluted affinitv purified antibodv) or 350 ⁇ g gioup (anti-CTB repeat specific, used 1/5 diluted anti-CIB antibodv)
  • the numbers in eacli group represent numbers of hamsters dead or alive 2 hr post-IP admmistiation of to.xin/antibodv mixture TABLE 29
  • N es denotes complete neutralization (05 dead) while no " denotes no neutiahzation (55 dead) ol toxin B 2 houis post-administration of mixture
  • Ihe pPB 1750-2360 antibody pool confers significant in vivo protection, equivalent to that obtained with the affinity purified CTB antibodies. This correlates with the observed high affinitv of this antibody pool (relative to the pMBl 750-2360 or pMB1970-2360 pools) as assayed bv Western blot analysis ( Figure 24).
  • toxin B neutralizing antibodies could be generated using specific recombinant toxin B proteins ( pPB l 750-2360) or specific adjuvants.
  • Antibodies raised against pMB l 750-2360 were capable of neutralizing the enterotoxin effect of toxin B w hen the recombinant protein was used to immunize hens in conjunction with the Gerbu adjuvant, but not when Freunds adjuvant was used.
  • toxin B-specific antibodies present in the neutralizing and non-neutralizing PI ' G preparations were isolated by affinity chromatography and tested for qualitativ e or quantitative differences.
  • IgY polycional antibodv preparations were affinity purified on the above column as follows The column was attached to an I 'V monitor (ISCO) and washed with PBS r ortv ml ahquots ol 2X PI G pieps (filter sterilized using a 045 ⁇ filter and quantified bv ()[),, before
  • the percentage ot toxin B repeat-specific antibodies present in each pieparation was determined using the quantifications ot antibodv yields trom the first column pass (amount ot
  • affinity- puri fied anti-toxin B repeat (aa 1 870-2360 of toxin B ) antibodies from the different preparations vvas administered to hamsters using the in vivo hamster model as described below .
  • the in vivo hamster model was utilized to assess the neutralizing ability of the affinity purified antibodies raised against ecombinant toxin B proteins purified in (a ) abov e.
  • a 4X IgY PEG preparation from a second independent immunization utilizing the pPB 1 750- 2360 antigen with Freunds adjuv ant vvas tested for in vivo neutralization The results are shown in fable 3 1 .
  • T able 3 1 demonstrate that: 1 ) as shown in Example 19 and reproduced here.
  • 1 .5 mg of affinitv purified antibody from pMBl 750-2360 immunized hens using Freunds adjuvant does not neutralize toxin B in vivo.
  • 300 ⁇ g of affinitv purified antibodv from similarly immunized hens utilizing Gerbu adjuvant demonstrated complete neutralization of toxin B in vivo.
  • Gerbu adjuvant in addition to enhancing the titer of antibodies reactive to the pMB l 750-2360 antigen relative lo Freunds adjuvant (demonstrated in ( a) above), also enhances the Teld of neutralizinu antibodies to this antigen, greater than 5 fold.
  • the amount of native toxin B added ( present as an addition to solubilizcd hamster stool ) to a given well is indicated (0 to 200 ng).
  • Antibody raised against the recombinant toxin B protein.
  • pMB 1 750- 2360(( ierbu). was affinity purified on the an affinity column containing pPB I 850-2360
  • toxin 1-5 protein and affinity-purified antitoxin can be used for the basis of a competitiv e immunoassay for the detection of toxin B in biological samples.
  • the assay can be used to detect samples containing less than 12.5 nu toxin A/well and as little as 50- 100 nu toxin B/well.
  • hese competitive inhibition assays demonstrate that native C difficile toxins and iecombinant ( difficile toxin proteins can compete for binding to antibodies raised against iecombinant ( ' difficile toxins demonstrating that these anti-recombinant toxin antibodies ptovi e effective diagnostic reagents
  • Affinity -purified antibodies against iecombinant toxin A oi toxin B were immobilized to 96 well microtiter plates as follo s
  • the wells were passively coated overnight at 4°C with affinity purified antibodies rai.scd against either pMA 1870-2680 (toxin A) or pMB1750- 2360(Gerbu) (toxin B).
  • the antibodies were affinity purified as described in Example 20.
  • the antibodies were used at a concentration of 1 ⁇ g/ml and 100 ⁇ l was added to each microtiter well. I he wells were then blocked with 200 ⁇ l of 0 5% BSA in PBS for 2 hours at room temperature and the blocking solution was then decanted.
  • Fhe plates were incubated for 2 hours at room temperature and then were w ashed three times w ith PBS.
  • the plates were incubated for another 2 hours at room temperature.
  • Fhe ' botulinum ty pe A neurotoxin gene has been cloned and sequenced [Thompson. ei al . Eur .1. Biochem. 1 89:73 ( 1990)]
  • the nucleotide sequence of the toxin gene is av ailable from the EMBL/GenBank sequence data banks under the accession number X52066: the nucleotide sequence of the coding region is listed in SEQ ID NO 27.
  • I he amino acid sequence of the ( ' hoiulinum ty pe A neurotoxin is listed in SEQ I D NO.28.
  • T he ty pe A neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds.
  • I he 50 kD carboxy- termmal portion of the heavy chain is referred to as the C fragment or the I I, domain.
  • lusion proteins comprising a synthetic C fragment gene derived from the C hoiulinum type A toxin and either a portion of the ( ' . difficile toxin protein or the MBP were constructed.
  • This example involved a) the construction of plasmids encoding C fragment fusion proteins and b) expression of C. botulinum C fragment fusion proteins in E. coh.
  • Example 1 Proteins in Example 1 1. it was demonstrated that the C difficile toxin A repeat domain can be efficiently expressed and purified in E. coli as either native (expressed in the pE ' F 23a vector in clone pPA 1 870-2680) or lusion (expressed in the pMALc vector as a fusion with the E. coli M BP in clone pMA I 870-2680) proteins. Fusion proteins comprising a fusion between the MBP. portions of the ( ' difficile toxin A repeat domain ( show n to be expressed as a soluble fusion protein ) and the C fragment of the C botulinum type A loxin were constructed. A fusion protein comprising the C fragment of the C hoiulinum type A toxin and the MBP was also constructed.
  • Figure 25 provides a schematic representation of the botulinal fusion proteins along w ith the donor constructs containing the C. difficile toxin A sequences or ( ' botulinum C Iragment sequences which were used lo generate the botulinal fusion proteins.
  • solid boxes represent C difficile toxin A gene sequences
  • the open boxes represent ( ' hoiulinum C fragment sequences
  • the solid black ovals represent the E. coli MBP.
  • FIG. 25 a restriction map of the pMA I 870-2680 and pPA l 100-2680 constructs ( described in E.xample 1 1 ) which contain sequences derived from the ( ' . difficile toxin A repeat domain are shown: these constructs were used as the source of C difficile toxin A gene sequences f r the construction of plasmids encoding fusions between the ( ' . hoiulinum C fragment gene and the ( ' difficile toxin A gene.
  • Fhe pMA 1870-2680 expression construct expresses high levels of soluble, intact fusion protein (20 mg/liter culture) which can be affinity purified on an amylose column ( purification described in Example 1 I d).
  • the pAlterBot construct ( Figure 25) was used as the source of C hoiulinum C fragment gene sequences for the botulinal fusion proteins.
  • pAlterBot was obtained from J. Middlebrook and R. Lemley at the U.S. Department of Defense.
  • pAlterBot contains a synthetic ( ' hoiulinum C fragment inserted in to the pALTER- l i 1 vector ( Promega).
  • This synthetic C fragment gene encodes the same amino acids as does the naturally occurring C I ragment gene.
  • the naturally occurring C fragment sequences like most clostridial genes, are extremely AT rich (Thompson et al.. supra). This high A/T content creates expression difficulties in E.
  • the nucleotide sequence of the ( ' hoiulinum C fragment gene sequences contained w ithin pAlterBot is listed in SEQ I D NO:22.
  • the first six nucleotides ( ATGGCT) encode a methionine and alanine residue, respectively. These two amino acids result from the insertion of the ( ' hoiulinum C fragment sequences into the pALTER® vector and provide the initiator methionine residue.
  • the amino acid sequence of the ( ' . hoiulinum C fragment encoded by the sequences contained within pAlterBot is listed in SEQ I D NO:23.
  • the first two amino acids ( Met Ala ) are encoded by vector-derived sequences. From the third amino acid residue onward ( Arg ). the amino acid sequence is identical to that found in the C botulinum type A toxm gene.
  • the botulinal gene sequences were removed from pAlterBot and were inserted into the pBluesc ⁇ pt plasmid (Stratagene) to generate pBIueBot (F igure 25).
  • pBIueBot was constructed as follows. Bacteria containing the pAlterBot plasmid were grown in medium containing tetracycline and plasmid DNA was isolated using the QlAprep-spm Plasmid Kit ( Qiagen ).
  • pAlterBot DNA w as digested w ith , ⁇ col and the resulting 3 " recessed sticky end was made blunt using the Klenow fragment of DNA polymerase I ( here after the Klenow fragment) I e pAlterBot DNA vvas then digested with Hindl ll to release the botulinal gene sequences ( the Bot insert) as a blunt ( filled Xcol site)- l/tndl l l fragment.
  • pBluescript vector DNA was prepared by digesting 200 ng of pBluesc ⁇ pt DNA w ith Smal and Hindl ll. fhe digestion products from both plasmids w ere resolved on an agarose gel.
  • Fhe pMABot clone contains a 2.4 kb insert deriv ed Irom the ( ' diffic ile tox A gene f used to the Bot insert ( i e. the C botulinum C fragment sequences deriv ed Irom pAlterBot) pMABot ( Figure 25) vvas constructed by mixing gel-purified DNA Irom ⁇ otll Hindlll digested pBIueBot ( the 1 .2 kb Bot fragment).
  • the pMCABot construct contains a 1 .0 kb insert derived from the ( ' difficile toxin A gene fused to the Bot insert (/ e. the ( ' hoiulinum C fragment sequences derived from pAlterBot)
  • pMCABot was constructed by digesting the pMABot clone with EcoRl to iemov e the 5 " end of the ( ' difficile toxin A repeat (see Figure 25.
  • the pMAI -c vector contains a EcoR] site 5 ' to the C difficile insert in the pMABot clone). Fhe restriction sites w ere filled and re gated together alter gel purification
  • the resultant clone (pMCABot. 1 igui e 25 ) generated an in-trame tusion between the MBP and the remaining 3 ' portion of the ( ' difficile tox A repeat domain fused to the Bot gene
  • I he pMNABot clone contains the 1 kb Spell Ec oRl (filled) fragment from the C diffic ile toxin A repeat domain (derived from clone pPA l 100-2680) and the 1.2 kb ( " hoiulinum C l i agmenl gene as a Xc ol ( filled)/////7c/III fragment (de ⁇ ved from pAlterBot)
  • I hese two liagments were inserted into the pMAL-c vector digested with XhallLhndlll
  • the two insert liagments were generated by digestion of the appropriate plasmid with EcoRl ( pPA l 100-2680) or ⁇ c ol ( pAlterBot) lollowed by treatment w ith the Klenow f i agment After treatment w ith the Klenow I ragment.
  • the plasmids were digested w ith the second en/yme (either Spel oi Hindll l ) All three fragments were gel purified, mixed and Prep-a-Genc purified prior to ligation f ollowing ligation and transformation, putative recombinants were analy zed by restriction analy sis, the EcoRl site was found to be regenerated at the f usion lunction. as w as predicted for a fusion between the filled EcoRl and ⁇ c ol sites
  • ⁇ construct encoding a tusion protein between the botuhnal ( f ragment gene and the MBP gene vvas constructed ( i e . this tusion lacks any C diffic ile toxin A gene sequences) and termed pMBot I he pMBot construct was made by removal of the ( ' difficile toxin A sequences from the pMABot construct and fusing the C Iragment gene sequences to the MBP This was accomplished by digestion of pMABot DNA with ⁇ 7//I (located in the pMALc poly lmkei 5 " to the Xbal site) and Xbal (located 3' to the hotl site at the toxA-Bot f usion junction ), filling in the Xbal site using the Klenow fragment, gel purifying the desired lestrietion f i agment.
  • I igure 26 the tollovving samples were loaded I anes I 6 contain protein purified liom / coh containing the pMAL -c pP ⁇ 1870-2680.
  • pMABot pMNABot pMC ⁇ Bot and pMBot plasmids respectively 1 ane 7 contains broad range moleculai weight protein markers (BioRad)
  • Ihe piotem samples were piepaied lor electrophoresis bv mixing ⁇ ⁇ l ol eluted piotein with 5 ⁇ l of 2X SDS-PAGE sample buffer (0125 mM Ins-HC I pll 68 2 mM EDI A 6% SDS 20% glvceiol 0025% bromophenol blue [i-mercaptoethanol is added to 5% betore use) Ihe samples were heated to 95°C for 3 mm.
  • T hese results demonstrate that high level expression of intact C hoiulinum C fragment ⁇ ' difficile toxin A fusion proteins in E. coli is feasible using the pMAL-c expression system. These results are in contrast to those reported by H. F. LaPenotiere. el al. ( 1 93 ). supra. I n addition, these results show that it is not necessary to fuse the botulinal C fragment gene to the (. ' . difficile toxin A gene in order to produce a soluble fusion protein using the pM ⁇ L-c system in / ' . coli.
  • the blots were blocked by incubation for 1 hr at 20°C in blocking buffer [ PBST ( PBS containing 0.1 % Tvveen 20 and 5% dry milk)].
  • PBST PBS containing 0.1 % Tvveen 20 and 5% dry milk
  • the blots were then incubated in 10 ml of a solution containing the primary antibody: this solution compri.sed a 1 /500 dilution of an anti-C hoiulinum toxin A IgY PEG prep (described in Example 3 ) in blocking buffer.
  • the blots were incubated for I hr at room temperature in the presence of the primary antibody.
  • Fhe blots were washed and developed using a rabbit anti-chicken alkaline phosphatase conjugate ( Boehringer Mannheim) as the secondary antibody as follows.
  • Fhe rabbit anti-chicken antibodv was diluted to 1 ⁇ g/ml in blocking buffer ( 10 ml final volume per blot ) and the blots were incubated at room temperature for I hour in the presence of the secondary antibody.
  • Fhe blots were then washed successiv ely with PBST. BBS-Tween and 50 mM Na.CO.. pH 9.5.
  • the blots were then developed in freshly-prepared alkaline phosphatase substrate buffer ( 100 ⁇ g/ml nitro blue tetrazolium.
  • a second set of 5 groups containing 3 CY female rats/group were immunized nasally or orally with one of the following combinations (4) 250 ⁇ g pMNABot protein per rat ( nasal and oral ) or 5) 250 ⁇ g pMAF-c protein per rat ( nasal and oral).
  • the fusion proteins were prepared for immunization as follows. The proteins ( in column buffer containing 10 mM maltose) were diluted in 0. 1 M carbonate buffer. pH 9.5 and administered orally or nasally in a 200 ⁇ l volume. The rats were lightly sedated with ether prior to administration. The oral dosing was accomplished using a 20 gauge feeding needle. The nasal dosing was performed using a P-200 micro-pipettor (Gilson).
  • Fhe rats were boosted 14 days after the primary immunization using the techniques described above and were bled 7 days later. Rats from each group were lightly etherized and bled from the tail. The blood was allowed to clot at 37°C for 1 hr and the serum was collected.
  • the serum from individual rats was analyzed using an ELISA to determine the anti-C. hoiulinum t pe A toxin IgG serum titer.
  • the ELISA protocol used is a modification of that described in Example 1 3c. Briefly. 96-well microtiter plates ( Falcon. Pro-Bind Assay Plates) were coated with ( ' . hoiulinum type A toxoid (prepared as described in Example 3a) by placing 100 ⁇ l volumes of ( ' . hoiulinum type A toxoid at 2.5 ⁇ g/ml in PBS containing 0.005% thimerosal in each well and incubating overnight at 4°C. The next morning, the coating suspensions were decanted and all wells were washed three times using PBS.
  • the mouse model is the art accepted method For detection of botulinal toxins in body fluids and for the evaluation of anti-botulinal antibodies [ F.J. Schantz and D.A. Kautter. J. ⁇ ssoc. Off. Anal. Chem. 61 :96 ( 1990 ) and I nvestigational New Drug ( BB-IND-3703) application by the Surgeon General of the Department of the Army to the Federal Food and Drug Administration ).
  • the anti-C hoiulinum type A toxin antibodies were prepared as follows.
  • Rats from the group given pMBot protein by nasal administration were boosted a second time with 250 ⁇ g pMBot protein per rat and serum was collected 7 days later. Serum from one rat from this group and from a preimmune rat w as tested for anti-C hoiulinum type A toxin neutralizing activity in the mouse neutralization model described below .
  • the circulating serum titer of the Co ⁇ naught preparation would be approximately 25 l U/ml assuming an average serum volume of 3 liters).
  • the circulating anti-C botulinum titer seen in rats nasally immunized with pMBot protein ( 168 l U/ml ) is 6.7 time higher than the necessary circulation titer of anti-C. botulinum antibodv needed to be protectiv e in humans.
  • Numbers represent the number of mice surviving at 72 hours which received serum taken from rats immunized with the pMBot protein.
  • mice survived but w ere sick after 72 hr.

Abstract

The present invention includes recombinant proteins derived from Clostridium botulinum toxins. In particular, soluble recombinant Clostridium botulinum type A, type B and type E toxin proteins are provided. Methods which allow for the isolation of recombinant proteins free of significant endotoxin contamination are provided. The soluble, endotoxin-free recombinant proteins are used as immunogens for the production of vaccines and antitoxins. These vaccines and antitoxins are useful in the treatment of humans and other animals at risk of intoxication with clostridial toxin.

Description

MULTIVALENT VACCINE FOR CLOSTRIDIUM BOTULINUM NEUROTOXIN
FIELD OF THE INVENTION
The present invention relates to the isolation ol polypeptides deπved from Clostridium boiulimtm neurotoxins and the use thereof as immunogens for the production of vaccines, including multivaient vaccines, and antitoxins.
BACKGROUND OF THE INVENTION
The genus Clostridium is comprised of gram-positive, anaerobic, spore-torming bacilli The natural habitat of these organisms is the environment and the intestinal tracts of humans and other animals. Indeed, clostridia are ubiquitous: they are commonly found in soil. dust, sewage, marine sediments.
Figure imgf000003_0001
c ,t,r P.H A Sneath et al . "Clostridium." Bcrgey s Manual * of Systematic
Figure imgf000003_0002
. Vol 2. pp 1 141 - 1200. Williams & \\ ilktns ( 1986).) Despite the identification ot approximate!) 100 species ol Closiridiu . only a small number have been recognized as etiologic agents of medical and merman, importance. Nonetheless, these species are associated w ith
Figure imgf000003_0003
serious diseases, including botulism, tetanus, anaerobic cellulitis. gas gangrene, bacteremia. pscudoincmbranous colitis, and clostridia! gastroenteritis Table 1 lists some of the species ot medical and importance and the diseases with which they are associated As virtual!) all of these species been isolated from fecal samples of apparent!} health) persons, some ol these isolates
Figure imgf000003_0004
be transient, rather than permanent residents ot the colomc flora
TABLE I lourtώiim Species Of Medical And Veterinary Importance*
Figure imgf000003_0005
TABLE 1
( loti idiu Species Of Medical And Veterinary Impoitance*
Figure imgf000004_0001
TABLE 1 i'losii idiun Species Of Medical And Veteπnaiv importance*
Figure imgf000005_0005
(. umpiled tiom P G Lngelkiik cl ul "Chnsif tuition". I'I maples and Piacine ot ( liniutl -Inuerohu Hue Mining, pp 22-23. Siai Publishing Co.. Belmont. CA (1992) I Stephen and R A Petrovvski. " losns II Inch
Figure imgf000005_0001
Memlvanes und Dei emulate Cells " in Ikiclci ml Toxins.2d ed . pp 66-67 Societv tor Mιcιobιolog> (1986). R Berko and A.I I letchei (eds I "Bacteria! Discuses."
Figure imgf000005_0002
16th ed . pp 116-1 6. Merck Reseaith Laboratories. Rahway. \ J (1992). and O II Sigmund and C M Eraser (eds ). "Clostridia! Infections " Merck I etennan Manual 5th ed . pp 396-409 Merck & Co . Rah ay NJ (1979)
In most cases, the pathogenicitv of these organisms is related to the release ol powerful exotoxms or liighl)
Figure imgf000005_0003
enzymes. Indeed, several species of the genus Closiridmni produce toxins and other enzymes of great medical and
Figure imgf000005_0004
significance. |C.L. Ilathevvav. Clin. Microbiol. Rev.3:66-98 (1990).)
Perhaps because of their significance for human and veterinary medicine, much research has been conducted on these toxins, in particular those of (' hoiulimnn and C difficile
C. botutimtm
Several strains of Closiridmni hot liniim produce toxins of significance to human and animal health. |C. . Hathevva). Clin. Microbiol. Rev.3.66-98 (I990)| The effects of these toxins range from diarrheal diseases that can cause destruction of the colon, lo paralytic effects that can cause death. Particular!) at risk for developing clostridial diseases are neonates and humans and animals in poor health (e _,' , those suffering from diseases associated with old age or lmmunodeficiencv diseases)
( Josii idi in botulinum pioduces the most poisonous biological toxm known 1 he lethal human dose is a mere 10 ' mg/kg bodv eight foi toxm in the bloodstream Botuhnal toxm blocks nerve transmission to the muscles lesulting in flaccid paralvsis When the toxin i caches anvvav and respiratorv muscles it results m respiratorv failure that can cause death |S Λinon I Infect Dis 154201-206(1986))
( hoiulinum spores aie carried bv dust and aie found on vegetables taken from the soil on liesh ints and on agricultural products such as honev I nder conditions lavoiable 0 to the oiganism the spores getminate to vegetative cells which pioduces toxm [S Ainon
Ann Rev ed 11541 (1980))
Botulism disease mav be giouped into lout tvpes based on the method ot introduction ot toxin into the bloodstream 1 ood-bome botulism results Irom ingesting πnproperlv pteseivcd and madequatelv heated food that contains botuhnal toxm there were "> -> cases ot lood-botne botulism in the United States between 1976 and 1984 | I Mac Dona Id c/ I
\m I I pidemiol 124794 (1986) ] The death rate due lo botuhnal toxin is 12"o and can be highei in paiticulai risk groups f( () 1 acket ei al Λm I Med 76794 (1984) ] Wound- induced botulism results liom C hoiulinum penetrating traumati/ed tissue and piυduung toxm that is absoibed into the bloodslieam Since 1950 thutv cases ot wound botulism have been 0 icpoited |\1 N S art/ Inaeiohic Span. I aiming Bacilli 1 In C loslitdia pp H-646 m
Ii D Davis <./ al (eds )
Figure imgf000006_0001
4th edition I B I ippincott C o (1990)] Inhalation botulism results when the tox is inhaled Inhalation botulism has been icported as the lesult ol accidental exposuie in the laboratorv |F llol/er Med Klin 41 1715 (|9()2)] and could anse it the toxin is used as an agent ol biological warlaie |I)R 1 tan/ ti al in Botulinum 5 and Tetanus
Figure imgf000006_0002
pp 471-476]
Infectious mlant botulism results liom ( hoiulinum colonization ot the infant intestine with pioduction ol toxin and its absoiption into the bloodslieam It is likelv that the bacteπum gains enliv when spores are ingested and subsequentlv germinate |S Arnυn I Infect Dis 1 4201 (1986) | There have been 500 cases repotted since it was first iccogni/ed in 1976
"U) |M N Swail/ supia \ Infant botulism strikes infants who are three weeks to eleven months old (greater than 90% of the cases are infants less than six months). [S. Arnon. J. Infect. Dis. 1 54:201 ( 1986). ] It is believed that infants are susceptible, due. in large part, to the absence of the full adult complement of intestinal microtlora. The benign microtlora present in the adult intestine provide an acidic environment that is not favorable to colonization by C. botulinum. Infants begin l ife with a sterile intestine which is gradually colonized by microflora. Because of the limited microtlora present in early infancy, the intestinal environment is not as acidic, allow ing for ( '. hoiulinum spore germination, growth, and toxin production. In this regard, some adults who have undergone antibiotic therapy which alters intestinal microtlora become more susceptible to botulism.
An additional factor accounting for infant susceptibility to infectious botulism is the immaturity of the infant immune system. The mature immune system is sensitized to bacterial antigens and produces protective antibodies. Secretory IgA produced in the adult intestine has the ability to agglutinate vegetative cells of ( '. hoiulinum. | S. Arnon. J. Infect. Dis. 1 54:201 ( 1 986). ] Secretory IgA may also act by preventing intestinal bacteria and their products from crossing the cells of the intestine. [S. Arnon. Epidemiol. Rev . :45 ( 1981 ). | The infant immune system is not primed to do this.
Clinical symptoms of infant botulism range from mild paralvsis. to moderate and severe paralv sis requiring hospitalization. to fulminant paralysis. leading to sudden death. f S. Arnon. Epidemiol. Rev. 3 :45 ( 1 98 1 ).]
The chief therapy for severe infant botulism is ventilatory assistance using a mechanical respirator and concurrent elimination of toxin and bacteria using cathartics, enemas, and gastric lavage. There were 68 hospitalizations in California for infant botulism in a single year with a total cost of over $4 million for treatment. [T.L. Frankovich and S. Arnon. West. .1. Med. 1 54: 103 ( ! 9 1 ). |
Different strains of Clostridium botulinum each produce antigenically distinct toxin designated by the letters Λ-G. Serotype A toxin has been implicated in 26% of the cases of food botulism; types B. E and F have also been implicated in a smaller percentage oϊ the food botulism cases [1 1. Sugiyama. Microbiol. Rev. 44:419 ( 1 980)]. Wound botulism has been reportedly caused by only types A or B toxins [H. Sugiyama. supra]. Nearly all eases of infant botulism have been caused by bacteria producing either type A or type B toxin. ( Exceptionally, one New Mexico case was caused by Closiridium hoiulinum producing type F toxin and another by Closiridium hoiulinum producing a type B-type F hybrid.) [S. Arnon. Epidemiol. Rev. 3 :45 ( 1981 ). ] Type C toxin affects waterfow l, cattle, horses and mink. Type D toxin affects cattle, and type E toxin affects both humans and birds. Λ trivalent antitoxin derived from horse plasma is commercially available from
Connaught Industries Etd. as a therapy for toxin types A. B. and E. However, the antitoxin has several disadvantages. First, extremely large dosages must be injected intravenouslv and/or intramuscularly. Second, the antitoxin has serious side effects such as acute anaphylaxis w hich can lead to death, and serum sickness. Final ly, the efficacy of the antitoxin is uncertain and the treatment is costly. ( CO. Tacket et al.. Am. .1. Med. 76:794
( 1984). I
A heptavalent equine botuhnal antitoxin which uses only the F(ab' )2 portion of the antibody molecule has been tested by the United States Military. ( M. Balad . I 'SAMRDC New sletter, p. 6 ( 1991 ). ] This w as raised against impure toxoids in those large animals and is not a high titer preparation.
A pentavalent human antitoxin has been collected from immunized human subjects for use as a treatment for infant botulism. The supply of this antitoxin is limited and cannot be expected to meet the needs of all indiv iduals stricken w ith botulism disease. I n addition, collection ot human sera must involve screening out HIV and other potentially serious human pathogens. ( P.J. Schwarz and S.S. Arnon. Western .1. Med. 1 56: 197 ( 1 992 ). j
Infant botulism has been implicated as the cause of mortality in some cases of Sudden I nfant Death Syndrome ( SI DS. also known as crib death). SI DS is officially recognized as infant death that is sudden and unexpected and that remained unexplained despite complete post-mortem examination. The link of SIDS to infant botulism came when fecal or blood specimens taken at autopsy from SI DS infants were found to contain C hoiulinum organisms and/or toxin in 3-4% of cases analyzed. [D.R. Peterson et al . Rev. Infect. Dis. 1 :630 ( 1979). ] In contrast, only 1 of 160 healthy infants (0.6%) had C. hoiulinum organisms in the leees and no botulinal toxin. ( S. Arnon et al.. Lancet, pp. 1 273-76. June 1 7. 1978. )
In dev eloped countries. SI DS is the number one cause of death in children between one month and one year old. ( S. Arnon et al.. Lancet, pp. 1 273-77. June 1 7. 1978.) More children die from SIDS in the first vear than from anv other simile cause of death in the First fourteen years of life. In the United States, there are 8.000-10.000 SIDS victims annually. Id
What is needed is an effective therapy against infant botulism that is free of dangerous side effects, is available in large supplv at a reasonable price, and can be salely and gently delivered so that prophv lactic application to infants is feasible.
Immunization of sublets with toxin preparations has been done in an attempt to induce linmunitv against botuhnal toxins A C hoiulinum vaccine comprising chemically inactivated (/ e . lormaldehyde-treated) ivpe A. B. C. D and E toxin is commercialh available for human usage However, this vaccine preparation has several disadvantages. First, the efficacy of this vaccine is variable (in particular, only 78% ol recipients produce protective levels ot anti-tvpe B antibodies following administration of the pπmarv series) Second, immunization is painful (deep subcutaneous inoculation is required lor administration), with adveise I factions being common (moderate to severe local reactions occur in approximatelv 6% ot recipients upon initial miection. this number rises to approximatelv I 1% of individuals who leceive booster lniections) [Informational Brochure tor the Pentavalent (ABCDE)
Botulinum l xoid. Centers lor Disease Control] Third, preparation oi the vaccine is dangeious as active tox must be handled
Figure imgf000009_0001
laboratory workers
What is needed are sate and eliective vaccine preparations loi administration to those at risk ol exposure to (' hoiulinum toxins
C. difficile
( difficile. Λ\Λ organism which gained its name due to dif iculties encountered in its isolation, has iccentlv been proven to be an etiologic agent ol diarrheal disease (Snealh et al . p 1165.) ( difficile is present in the gastrointestinal li act ot approximatelv 3% ol health} adults, and 10-30% of neonates without adverse effect (Swart/, at p.644): by other estimates. C difficile is a part of the normal gastrointestinal flora of 2-10% ot humans. [G F Brooks el al . (eds.) "Infections ( aused h\ Anaerobic Bacteria." Jcneetz Melntck el s Medical Microbiology. 19th ed . pp.257-262. Appleton & Lange. San Mateo. CΛ (1991) I As these organisms are relatnelv resistant to most commonlv used antimicrobials. when a patient is treated with antibiotics, the other members ot the normal gastrointestinal flora are suppressed and C. difficile flourishes, producing cytopathic toxins and enterotoxins. It has been found in 25% of cases of moderate diarrhea resulting from treatment with antibiotics, especially the cephalosporins. clindamycin. and ampiciilin. [M.N. Swartz at 644.] Importantly. C. difficile is commonly associated w ith nosocomial infections. The organism is often present in the hospital and nursing home environments and may be carried on the hands and clothing of hospital personnel who care for debilitated and immunocompromised patients. As many of these patients are being treated w ith antimicrobials or other chemotherapeutic agents, such transmission of ( '. difficile represents a significant risk factor for disease. ( Engelkirk et al.. pp. 64-67.) C. difficile is associated with a range of diarrhetic il lness, ranging from diarrhea alone to marked diarrhea and necrosis of the gastrointestinal mucosa w ith the accumulation of inflammatory cells and fibrin, which forms a pseudomembrane in the affected area. ( Brooks el al. ) I t has been found in ov er 95% of pseudomembranous enterocolitis cases. ( Swartz. al p. 644. ) This occasionally latal disease is characterized by diarrhea, multiple small colonic plaques, and toxic megacolon. ( Swartz. at p. 644. ) Although stool cultures are sometimes used for diagnosis, diagnosis is best made by detection of the heat labile toxins present in fecal Filtrates from patients with enterocolitis due to ( '. difficile. ( Swarlz. at p. 644-645: and Brooks ei al.. at p. 260.) ( ' difficile toxins are cytotoxic tor tissue/cell cultures and cause enterocolitis when injected intraeecally into hamsters. ( Swartz. at p. 644. ) The enterotoxicity oϊ C. difficile is primarily due to the action of two toxins. designated A and B. each of approximately 300.000 in molecular weight. Both are potent cytotoxins. with toxin A possessing direct enterocytoloxic activity. ( I .yerly ei al . I nfect. Immun. 60:4633 ( 1992). | Unlike toxin A of (.'. perfrin ens. an organism rarely associated w ith antimicrobial-associated diarrhea, the toxin of ( '. difficile is not a spore coat constituent and is not produced during sporulation. ( Swartz. at p. 644. ) ( ' difficile toxin A causes hemorrhage, fluid accumulation and mucosal damage in rabbit ileal loops and appears to increase the uptake of toxin B by the intestinal mucosa. Toxin B does not cause intestinal fluid accumulation, but it is 1000 times more toxic than toxin A to tissue culture cells and causes membrane damage. Although both toxins induce similar cellular effects such as actin disaggregation. differences in cell specificity occurs. Both toxins are important in disease. [Borriello et al . Rev. Infect. Dis.. I 2(suppl. 2):S 185 ( 1990); Lyeriy ei al.. Infect. Immun.. 47:349 ( 1985): and Rolfe. Infect. Immun.. 59: 1 223 ( 1990). ] 'Foxin A is thought to act first by binding to brush border receptors, destroying the outer mucosal layer, then allowing toxin B to gain access to the underlying ' 5 tissue. These steps in pathogenesis would indicate that the production of neutralizing antibodies against toxin A may be sufficient in the prophylactic therapy of CDAD. However, antibodies against toxin B may be a necessary additional component for an effective therapeutic against later stage colonic disease. Indeed, it has been reported that animals require antibodies to both toxin A and toxin B to be completely protected against the disease. 10 I Kim and Rol fe. Λbstr. Ann. Meet. Am. Soc. Microbiol.. 69:62 ( 1987).]
( difficile has also been reported to produce other toxins such as an enterotoxin different from toxins A and B [ Banno l al.. Rev. Infect. Dis.. 6(Suppl. 1 :S 1 1 -S20 ( 1984)|. a lo molecular w eight toxin | Rihn ei al.. Biochem. Biophys. Res. Comm.. 1 24:690-695 I I 984 ) |. a motility altering factor [Justus el al.. Gastroenterol.. 83 :836-843 ( 1 982 ) ]. and 1 5 perhaps other toxins. Regardless. ( ' difficile gastrointestinal disease is of primary concern.
It is significant that due to its resistance to most commonly used antimicrobials. C. difficile is associated w ith antimicrobial therapy with virtually all antimicrobial agents ( although most commonly ampiciilin. clindamycin and cephalosporins). It is also associated w ith disease in patients undergoing chemotherapy with such compounds as methotrexate. 5- 20 lluorouracil. cyclophosphamide. and doxorubicin. [ S.M. Finegold el al.. Clinical iiuide lo
Anaerobic Infections, pp. 88-89. Star Publishing Co.. Belmont. CA ( 1992). |
'Frealment of ( ' difficile disease is problematic, given the high resistance of the organism. Oral metronidazole. bacitracin and vancomycin have been reported to be effective. ( Finegold ei al . p. 89. ) However there are problems associated with treatment utilizing these 25 compounds. Vancυmycin is v ery expensive, some patients are unable to take oral medication, and the relapse rate is high (20-25%). although it may not occur for several weeks. Id.
( '. difficile disease would be prevented or treated by neutralizing the effects of these toxins in the gastrointestinal tract. Thus, what is needed is an effective therapy against C. difficile toxin that is free of dangerous side effects, is available in large supply at a reasonable price, and can be safely delivered so that prophylactic application to patients at risk of developing pseudomembranous enterocolitis can be effectively tieated.
DESCRIPTION OF THE DRAWINGS I igure 1 shows the reactivity of anti-C hoiulinum IgY by Western blot
I igure 2 shows the IgY antibody titer to (' hoiulinum type A toxoid in eggs, measured bv 1 I ISA
I igure 3 shows the results of C difficile toxm A neutralization assav s
Figure 4 shows the results oi (' difficile toxin B neutralization assav s I igure 5 shows the results oϊ C difficile toxin B neutralization assays
I iguie 6 is a lestπction map ot C difficile tox A gene, showing sequences ol primers 1-4 (SEQ ID NOS- 1-4)
I igure 7 is a Western blot ol (' difficile tox A leaclive protein
I igure 8 shows ( difficile toxm A expression constructs. I igure 9 shows C difficile toxin A expression constructs
1 igure 10 shows the purification ot recombinant (' difficile tox A
1 igure 11 shows the results ol (' difficile toxm A neutiah/ation assav s with antibodies reactive lo teeombinant toxm A
I iguie 12 shows the results for a (' difficile toxm A neutralization plate I iguie 13 shows the results for a C difficile toxin A neutralization plate
1 iguie 14 shows the results ot recombinant C difficile toxin A neutralization assav s
I iguie 15 shows ( difficile toxin A expression constructs
I iguie 16 shows a chromatograph plotting absorbance at 280 nm against letention time loi a pMAl 870-680 IgY PI (i preparation. I igure 17 shows two recombinant (' difficile toxm B expression constructs figuie 18 shows (' difficile toxin B expression constructs
Figure 19 shows (' difficile toxm B expression constructs
I igure 20 shows (' difficile toxin B expression constructs
I iguie 21 is an SDS-PAGE gel showing the purification ol recombinant (' difficile toxm B lu ion protein 1 iguie 22 is an SDS-PAGE gel showing the purification of two histidme-tagged recombinant C difficile toxin B proteins
I igure 23 shows ( difficile toxin B expression constructs
1 igure 24 is a Western blot ol ( difficile toxin B reactive piotem Figure 25 shows ( hoiulinum t pe A toxin expression constructs constructs used to piovidc ( hoiulinum or ( difficile sequences are also shown
Figuie 26 is an SDS-PAGF gel stained with Coomaisse blue showing the purification ol iccombinant ( botulinum tvpe A toxin fusion proteins
Figuie 27 shows ( botulinum tv pc A toxm expression constructs constiucts used to piovidc ( hoiulinum sequences are also shown
I iguie 28 is an SDS-PAGT gel stained with ( oomaisse blue showing the purification of pIlisBot piotetn using the Ni-NIΛ lesin
I iguie 29 is an SF)S-PΛGE gel stained with Coomaisse blue showing the expression of pIlisBot protein in BI 21(1X3) and BF21(DF 3)pl vsS host cells I igme 30 is an SDS-PAGF gel stained with Coomaisse blue showing the purification ol pliisBol piotem using a batch absorption procedure
1 iguie 31 is an SDS-P \GE gel stained with Coomaisse blue show inn the puiillcation ol pliisBol and pllιsBot(natιve) pioteins using a Ni-N I \ column
I igure 32 is an SDS-PAGI gel stained with Coomaisse blue show mi: the purification ol pllisBolΛ protein expressed in pllιsBotA(svn) kan laclq 17/p C YC Gro/BI 2KDE3) cells using an IDA column
I iguie 33 is an SDS-PAGF gel stained with C omaisse blue showing the purification ol pHisBot \ pHisBotB and pllisBotE proteins bv IDΛ chromatographv followed bv chiomatogiaphv on S-100 to lemove tolding chaperones I igure 34 is an SDS-PAGE gel stained with Coomaisse blue showing the extracts derived tiom pHisBotB amp T7lac/BL21(Dr3) cells betoie and after purification on a Ni- NΪA column
Figure 35 is an SDS-PAG1 gel mn under native conditions and stained with C oomaisse blue showing the removal ol folding chaperones tiom IDA-purified BotB protein usirm a S-1 0 column Figure 36 is an SDS-PAGE gel stained with Coomaisse blue showing proteins that eluted during an imidazole step gradient applied to a IDA column containing a lysate of pHisBotB kan laclq T7/pACYCGro/BL21 (DE3) cells.
Figure 37 is an SDS-PAGE gel run under native conditions and stained w ith Coomaisse blue showing IDA-purified BotB protein before and after ultrafiltration.
Figure 38 is an SDS-PAGE gel stained with Coomaisse blue showing the purification of BotE protein using a NiNTA column.
F igure .39 is an SDS-PAGE gel stained with Coomaisse blue showing extracts derived from pHisBotA kan 17 lac/BL21 (DE3) pLysS cells grown in fermentation culture. F igure 40 is a chromatogram show ing proteins present after I DA-purified BotE protein was applied to a S- 100 column.
DEFINITIONS
To facilitate understanding of the invention, a number of terms are defined below . As used herein, the term "neutralizing" is used in reference to antitoxins, particularly antitoxins comprising antibodies, w hich have the ability to prevent the pathological actions of the toxin against which the antitoxin is directed.
As used herein, the term "overproducing" is used in reference to the production of clostridial toxin polypeptides in a host cell and indicates thai the host cell is producing more of the clostridial toxin by virtue of the introduction of nucleic acid sequences encoding said clostridial toxin polypeptide than would be expressed by said host cell absent the introduction of said nucleic acid sequences. To allo ease of purification of toxin polypeptides produced in a host cel l it is preferred that the host cell express or overproduce said toxin polypeptide at a level greater than 1 mg/liter oϊ host cell culture. "A host cell capable of expressing a recombinant protein at a level greater than or equal to 5% of the total cellular protein" is a host cell in which the recombinant protein represents at least 5% of the total cellular protein. To determine what percentage of total cellular protein the recombinant protein represents, the following steps are taken. A total of 10 C)DM„, units of recombinant host cel ls (e tc.. 200 μl of cells at OF),,,,, 50/ml ) are removed (at a timepoint known to represent the peak of expression of the desired recombinant protein) to a 1 .5 ml microfuge tube and pelleted for 2 min at maximum rpm in a microfuge. The
1 7 - pellets are resuspended in 1 ml of 50 mM NaHPO.,, 0.5 M NaCl. 40 mM imidazoie buffer ( pH 6.8) containing 1 mg/ml lysozyine. The samples are incubated for 20 min at room temperature and stored ON at -70°C. Samples are thawed completely at room temperature and sonicated 2 X 1 0 seconds w ith a Branson Sonifier 450 microtip probe at # 3 power setting. The samples are centrifuged for 5 min. at maximum rpm in a microfuge. An aliquot
( 20 μl ) of the protein sample is removed to 20 μl 2X sample buffer (this represents the total protein extract). The samples are heated to 95°C for 5 min. then cooled and 5 or 10 μl are loaded onto 1 2.5% SDS-PAGE gels. High molecular weight protein markers are also loaded to allow for estimation of the MW of identified recombinant proteins. After electrophoresis. protein is detected generally by staining with Coomassie blue and the stained gel is scanned using a densitometer to determine the percentage of protein present in each band. In this manner, the percentage of protein present in the band corresponding to the recombinant protein of interest may be determined. It is not necessary that Coomassie blue be employed for the detection of protein, a number of fluorescent dyes \e.χ.. Sypro orange S-665 1 ( Molecular Probes. Eugene. OR] may be employed and the stained gel scanned using a lluoroimager | c.,t,'.. Fluor I mager SI ( Molecular Dynamics. Sunnyvale. CA ) |.
"A host cell capable of expressing a recombinant protein as a soluble protein at a level greater than or equal to 0.25% of the total soluble cellular protein" is a host cell in which the amount of soluble recombinant protein present represents at least 0.25% of the total cellular protein. As used herein "total soluble cellular protein" refers to a clarified PEI lysate prepared as described in Example l (c )( iv). Briefly, cells are harvested follow ing induction of expression oϊ recombinant protein ( at a point of maximal expression ). The cells are resuspended in cell resuspension buffer (CRB: 50 mM NaPO,. 0.5 M NaCl. 40 M imidazoie. pl-f 6.8) to create a 20% cell suspension (wet weight of cells/volume of CRB ) and cell lysates are prepared as described in Example 31 (c)(iv) {i.e.. sonication or homogenization followed by centrifugation). The cell lysate is then flocculated utilizing polyethyleneimine ( PEI) prior to centrifugation. PEI (a 2% solution in dPFO. pH 7.5 with I ICI) is added to the cell ly sate to a final concentration of 0.2%. and stirred for 20 min at room temperature prior to centrifugation [ 8.500 rpm in JA I 0 rotor ( Beckman ) for 30 minutes at 4°C' |. This treatment removes RNA. DNA and cell wall components, resulting in a clarified, low viscosity ly sate
( "PEI clarified lysate"). The recombinant protein present in the PEI clarified ly sate is then purified {e.g., by chromatography on an IDA column for his-tagged proteins). The amount of purified recombinant protein (i. e.. the eluted protein) is divided by the concentration of protein present in the PL- I clarified lysate (typically 8 mg/ml when using a 20% cell suspension as the starting material ) and multiplied by 1 00 to determine what percentage of total soluble cellular protein is comprised of the soluble recombinant protein ( see Example
33b).
As used herein, the term "fusion protein" refers to a chimeric protein containing the protein of interest (i. e.. C. hoiulinum toxin A. B. C. D. E. !■'. or G and fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of a non-toxin protein). The fusion partner may enhance solubility of the C. hoiulinum protein as expressed in a host cell, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both. I f desired, the fusion protein may be remov ed from the protein of interest (i. e.. toxin protein or fragments thereof) prior to immunization by a variety of enzymatic or chemical means known to the art. As used herein the term "non-toxin protein" or "non-toxin protein sequence" refers to that portion of a fusion protein which comprises a protein or protein sequence w hich is not deriv ed from a bacterial toxin protein.
The term "protein of interest" as used herein refers to the protein whose expression is desired w ithin the fusion protein, in a fusion protein the protein of interest w ill be joined or fused with another protein or protein domain, the fusion partner, to allow for enhanced stabilit of the protein of interest and/or ease of purification of the fusion protein.
As used herein, the term "maltose binding protein" refers to the maltose binding protein oϊ /•.'. coli. A portion of the maltose binding protein may be added to a protein of interest lo generate a fusion protein: a portion of the maltose binding protein may merely enhance the solubility of the resulting fusion protein when expressed in a bacterial host. On the other hand, a portion of the maltose binding protein may allo affinity purification of the fusion protein on an amylose resin.
As used herein, the term "poly-histidine tract" when used in reference to a fusion protein refers to the presence of two to ten histidine residues at either the amino- or carboxy- terminus of a protein of interest. A poly-histidine tract oϊ six lo ten residues is preferred.
The poly -histidine tract is also defined functionally as being a number of consecutive histidine residues added to the protein of interest which allows the affinity purification of the resulting fusion protein on a nickel-chelate or IDA column
\s used herein, the term 'purified" or "to puπfv" icters to the removal ot contaminants Irom a sample I or example, antitoxins are purified bv lemoval of contaminating non-immunoglobuhn proteins, they aie also purified bv the removal ot immunoglobulin that does not bind toxin The removal ot non-immunoglobuhn proteins and/oi the removal oi immunoglobuhns that do not bind toxin results m an increase in the percent ot toxm-ieacti e immunoglobuhns in the sample In another example recombinant toxm polvpeptides are expressed in bacterial host cells and the toxm polvpeptides are purified bv the removal oi host ceil pioteins the percent ot recombinant toxin polypeptides is thereby incieased in the sample \ddιtιonallv the recombinant toxin polypeptides aie punfied bv the icmoval ol host cell components such as lipopolv saccharide (c t,' endotoxin)
I he teπn lecombmant DNA molecule" as used herein reteis to a D Λ molecule which is comprised ol segments of DNA joined together bv means oi molecular biological techniques
I he teim 'lecombmant piotem or "lecombmant polv peptide' as used heie ieleis to a piotem molecule which is expiessed Irom a recombinant DNA molecule
I he term 'native protein' as used herein refers to a protein which is isolated from a natuial souice as opposed to the pioduction of a protein bv iecombinant means \s used herein the teim portion' when in refeience to a piotem (as in 'a portion ol a given piotem") teteis to liagments ol that protein The tiagments mav range m size Irom loin am o acid tesidues to the entire ammo acid sequence minus one ammo acid
\s used herein 'soluble when in reference to a protein pioduced bv iecombinant DNA technology a host cell is a piotem which exists in solution in the cvtoplasm of the host cell, it the piotem contains a signal sequence the soluble protein is exported to the penplasmic space in bactcπa hosts and is secreted into the cultuie medium in eucaryotic cells capable ol secretion oi bv bacterial host possessing the appropriate genes (/ e . the k gene) In contiast. an insoluble piotem is one which exists in denatured foim inside cv toplas ic granules (called inclusion bodies) in the host cell High level expression (/ c greater than 10- 20 mg iecombinant piotem/htei ot baetenal culture) ot tecombinani pioteins often lesults m the expiessed protein being lound in inclusion bodies in the baetenal host cells \ soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.
A distinction is drawn between a soluble protein ( i e . a protein which w hen expressed in a host cell is produced in a soluble form) and a "solubihzed" protein An insoluble i ecombinant protein found inside an inclusion body may be solubihzed (/ e . rendered into a soluble form ) by treating purified inclusion bodies with denaturants such as guamdine hy drochlonde. urea or sodium dodecy I sulfate ( SDS ) I hese denaturants must then be i cmov ed from the solubihzed protein preparation to allow the recovered protein to renature ( retold) Not all proteins will refold into an active conformation alter solubihzation in a denaturant and removal of the denaturant. Many proteins precipitate upon remov al of the denaturant SDS may be used to solubihze inclusion bodies and w ill maintain the proteins in solution at low concentration Howev er, dialysis w ill not alway s remove all ol the SDS ( SDS can l rm micelles w hich do not dialy ze out); therefore. SDS-solubih/ed inclusion body protein is soluble but not refolded
A distinction is drawn between proteins which are soluble ( i e . dissolv ed ) in a solution dev oid ol significant amounts ot ionic detergents ( c tc . SDS ) or denaturants (c ,g . ui ea. guunidme hy drochloride) and proteins which exist as a suspension ol insoluble protein molecules dispersed within the solution A soluble protein will not be removed Irom a solution containing the protein by centrifugation using conditions suf ficient to remov e bacteria present in a liquid medium ( i e . centrif ugation at 1 2.000 \ g tor 4-5 minutes ) I oi example, to test vv hethei two proteins, protein A and protein B. ai e soluble m solution, the tw o proteins ai e placed into a solution selected f rom the group consisting of PBS-NaC 1 ( PBS containing 0 5 M NaCl ). PBS-NaCI containing 0.2% I ween 20. PBS. PBS containing 0 2% 1 ween 20. PBS-C ( PBS containing 2 mM CaCF). PBS-C containing either 0 1 or 0 5 % Tween 20. PBS-
C containing either 0 1 or 0.5% NP-40. PBS-C containing either 0 1 oi 0 5% Tπton X- 1 00. PBS-C containing 0 1 % sodium deoxy cholate The mixture containing proteins A and B is then eenti ifuged at 5000 \ g for 5 minutes I he supernatant and pellet lormed by centrifugation are then assayed loi the presence ot protein A and B I I protein A is found m the supernatant and not in the pellet [except for minor amounts ( i e . less than 10%) as a result ol trapping], piotem is said to be soluble in the solution tested I I the majonty ot piotem B is found in the pellet (/ e . greater than 90%). then protein B is said to exist as a suspension in the solution tested
As used heiein the term theiapeutic amount' leteis to that amount ot antitoxin lequned to neutralize the pathologic effects of one oi moie clostridial toxins in a sub|ect Ihe teim 'pvrogen as used herein refers to a fc ei -producing substance Pvrogens mav be endogenous to the host (c g prostaglandins) oi mav be exogenous compounds (e tc bacteria] endo- and exotoxins nonbacteπal compounds such as antigens and certain steroid compounds etc ) Ihe presence ol pvrogen in a pharmaceutical solution mav be detected using the I S Pharmacopeia (USP) rabbit fever test (United States Pharmacopeia. Vol XXII ( 1990) United States Pharmacopeial C onvention Rockville MD p 151)
I he teim endotoxin" as used herein refers to the high moleculai weight complexes associated with the outer membrane ot gram-negati e bactciia Unpunfied endotoxin contains lipids pioteins and carbohvdiates Highlv purified endotoxin does not contain piotem and is icleiied to as hpopolv saccharide (EPS) Because unpunfied endotoxin is of concern in the production ol pharmaceutical compounds (eg. proteins produced in t coli using iecombinant
DNA technologv ) the teim endotoxin as used heiein refers to unpunfied endotoxin Bacterial endotoxin is a well known pvrogen
\s used herein the teim endotoxm-free when used in leterence to a composition to be admmisteicd parenterallv (with the exception of intrathecal administration) to a host means that the dose to be dehveied contains less than 5 F U/kg bodv weight [I DA Guidelines lor
Paientcial Diugs (December 1987)]
Figure imgf000019_0001
a weight ol 70 kg tor an adult human the close must contain less than 350 I U lo meet IDA Guidelines lot parcnteral administration 1 ndotoxin levels are measured herein using the I ltnulus Amebocvte 1 v sale (1 ΛI ) test (1 imulus Amebocvte 1 vsate Pv ιochιomeI Associates of Cape C d Inc Woods Hole MA) lo mcasuie endotoxin levels in preparations ol recombinant proteins.05 ml ot a solution comprising 05 mg ol purified iecombinant protein in 50 mM NaPO, pH 70 03M NaCl and 10% glvceiol is used in the I AL assav according to the manutactuier s instructions for the endpoint chiomogenic without diazo-couphng method [the specific components ol the buffei containing iecombinant piotem to be analvzed m the 1 ΛF test aie not impoitant unv buffei having a neutial pH mav be emplovcd (see tor example alternative butfeis emploved in
F xamples 34 40 and 45)| Compositions containing less than oi equal to than 25() endotoxin units (EU)/mg of purified recombinant protein are herein defined as "substantially endotoxm- free." Preferably the composition contains less than or equal to 100. and most preferably less than or equal to 60. (EU)/mg of purified recombinant protein Typically, administration ot bacterial toxins or toxoids to adult humans for the purpose ol vaccination involves doses of about 10-500 μg protein/dose I herefore. administration of 10-500 μg of a purified recombinant protein to a 70 kg human, wherem said purified recombinant protein preparation contains 60 EU/mg protein, results in the introduction of only 0.6 to 30 EU (/ e..0.2 to 8.6% ol the maximum allowable endotoxin burden per parenterai dose). Administration of 10-500 μg of a purified recombinant protein to a 70 kg human, wherein said purified recombinant protein preparation contains 250 EU/mg protein, results in the introduction of only 2.5 to 125
EU (/ <.'..07 to 36% ol' the maximum allowable endotoxin burden per parenterai dose)
The L M test is accepted by the U S. FDA as a means of detecting bacterial endotox s (21 Cl R. ^ 660.100 -105). Studies have shown that the LΛE test is equivalent oi superior to the USP rabbit pvrogen lest for the detection of endotoxin and thus the 1 Al. test can be used as a surrogate for pyrogenicity studies in animals [F C Perason. P\rogens endoioxins I AL testing and
Figure imgf000020_0001
Marcel Dekker. New York (1985). pp 150-!55|. Ihe l-DΛ Bureau of Biologies accepts the LΛL assay in place ot the USP rabbit pvrogen test so long as the LΛE assay utilized is shown to be as sensitive as. or more sensitive as the rabbit test |I ed Reg..38.26130 (1980)|. Ihe term "mυnovalent" when used in reference to a clostridial vaccine reters to a vaccine which is capable ol provoking an immune response in a host animal duected against a single type of clostridial toxm For example, if immunization ot a host with (' hoiulinum type A toxin vaccine induces antibodies in the immunized host which protect against a challenge with t pe A toxin but not against challenge with ty e B. C D. E. F or G toxins. then the type A vaccine is said to be monovalent. In contrast, a "muluvaleπt" vaccine provokes an immune response in a host animal directed against several (/ e . more than one) clostridial toxins. I or example, if immunization of a host with a vaccine comprising (' botulinum type A and B toxins induces the production of antibodies which protect the host against a challenge with both type A and B toxin, the vaccine is said to be multivalent (in particular, this hypothetical vaccine is bivalent) \.s used heiein the term "lmmunogenicallv-effective amount" refers to that amount ot an immunogen required to invoke the production of protecti e levels ol antibodies m a host upon vaccination
The teim 'piotective level when used in reference to the le el of antibodies induced upon immunization of the host with an immunogen which comprises a bacterial toxin means a level of ciiculating antibodies sufficient to protect the host Irom challenge with a lethal dose of the toxin
\s used heiein the teims protein' and "polvpeptide letei to compounds comprising amino acids |omed via peptide bonds and are used interchangeablv The teims toxin and "neurotoxin" when used m leterence to toxins produced bv members (/ c species and stiains) of the genus ( lostndium aie used interchangeablv and telei lo the proteins which are poisonous to nerve tissue
Ihe teim leceptoi-binding domain" when used in retcrencc to a ( botulinum toxin icteis to the caiboxv-teiminal portion ot the heavv chain (H( or the C liagment) of the toxin which is piesumed to be icsponsible tor the binding ol the active toxin (/ c the derivative toxm compπsing the H and I chains )oιned via disullide bonds) to leceptois on the surface ol svnaptosomes The receptor-binding domain tor C hoiulinum tvpe \ toxin is defined heiem as compiismg amino acid lesidues 861 thiough 1296 ol SI Q ID NO 28 Ihe icceptoi- binding domain for ( hoiulinum tvpe B toxin is defined herein as comprising amino acid lesidues 848 thiough 1291 ol STQ ID NO 40 (strain rklund I7B) Ihe receptoi-bindmg domain ol ( botulinum tvpe C 1 toxin is defined heiein as comprising ammo acid lesidues 856 thiough 1291 ot SF Q ID NO 60 Ihe receptor-binding domain ol C hoi ul mum tvpe D toxin is defined heiem as comprising amino acid lesidues 852 thiough 1276 o! SI Q ID NO 66 Ihe leceptor-binding domain ol C hoiulinum tvpe F toxin is defined herein as comprising ammo acid lesidues 835 thiough 1250 ot SF Q ID NO 50 (Beluga stiain) Ihe leceptoi -binding domain ot C hoiulinum tvpe i toxin is defined herein as comprising ammo acid lesidues 853 through 1274 ot SEQ ID NO 71 Ihe receptor-binding domain ot ( botulinum tvpe CJ toxm is defined herein as comprising ammo acid residues 853 thiough 1297 ol STQ ID NO 77 Within a given serotvpe small variations in the pπmarv amino acid sequence ol the botuhnal toxins isolated Irom different stiains has been repotted [Whelan ti al. ( 1992). supra and Minton ( 1995) Curr. Top. Microbiol. Immunol. 195: 161 - 194], The present invention contemplates fusion proteins comprising the receptor-binding domain of C. hoiulinum toxins from serotypes A-G including the variants found among different strains within a given serotype. I he receptor-binding domains listed above are used as the prototype for each strain within a serotype. Fusion proteins containing an analogous region from a strain other than the prototype strain are encompassed by the present invention.
F usion proteins comprising the receptor binding domain (i.e.. C fragment ) of botuhnal toxins may include amino acid residues located beyond the termini of the domains defined above. For example, the pFlisBotB protein contains amino acid residues 846- 1291 of SEQ ID O:40; this fusion protein thus comprises the receptor-binding domain for C. hoiulinum type
B toxin as defined above ( i. e.. I le-848 through Glu- 1291 ). Similarly. pHisBotE contains amino acid residues 827- 1 252 of SEQ I F) NO:50 and pHisBotG contains amino acid residues 85 1 - 1 297 of SEC) I D NO: 77. Thus, both pHisBotE and pHisBotG fusion proteins contain a few amino acids located beyond the N-terminus of the defined receptor-binding domain. The terms "native gene" or "native gene sequences" are used to indicate DNA sequences encoding a particular gene which contain the same DNA sequences as found in the gene as isolated from nature. In contrast, "synthetic gene sequences" are DNA sequences w hich are used to replace the naturally occurring DNA sequences w hen the naturally occurring sequences cause expression problems in a giv en host cell. For example, naturally- occurring DNA sequences encoding codons which are rarely used in a host cell may be replaced (e.g.. by site-directed mutagenesis) such that the synthetic DNA sequence represents a more frequently used codon. The native DNA sequence and the synthetic DNA sequence w ill pref erably encode the same amino acid sequence.
SUMMARY OF THE INVENTION
The present invention relates to the production of polypeptides derived from toxins particularly in recombinant host cells. In one embodiment, the present invention provides a host cell containing a recombinant expression vector, said vector encoding a protein comprising at least a portion of a Closiridium hoiulinum toxin, said toxin selected from the group consisting oϊ type B toxin and type E toxin. The present invention is not limited by the nature of sequences encoding portions of the ( '. hoiulinum toxm. These sequences may be derived irom the native gene sequences or alternatively thev may comprise synthetic gene sequences Synthetic gene sequences aie emploved when expression of the native gene sequences is problematic in a given host cell (e g, when the native gene sequences contain sequences resembling yeast transcription termination signals and the desired host cell is a veast cell)
In one embodiment the host cell is capable of expressing the recombinant C hoiulinum toxin protein at a level gieatet than oi equal to 2% to 40% of the total cellular piotem and preleiably at a level gteater than or equal to 5% of the total cellular protein In another embodiment the host cell is capable of expressing the recombinant ( hoiulinum toxm piotem as a soluble protein at a level greater than oi equal to 025% of the total cellulai piotem and pielerablv at a level greater than or equal to 025% to 10% ot the total cellular protein
Ihe present invention is not limited bv the natute ot the host cell emploved foi the pioduction ol iecombinant C hoiulinum toxm pioteins In a prefeired embodiment the host cell is an / coli cell In another pieterred embodiment, the host cell is an insect cell, paiticulailv pieterred insect host cells are Spodopieia fiugipeida (Sf9) cells In anothei pielened embodiment the host cell is a veast cell particularlv pieterred veast cells are Pic/va pashms cells in anothei embodiment the invention provides a host cell containing a iecombinant expiession said vectoi encoding a fusion protein comprising a non-toxm protein sequence and at least a portion of a C losit idiuni hoiulinum toxm. said toxin selected tiom the moup consisting ol tv e B toxm and t pe E toxin The invention is not limited bv the nature ol the poition ol the C losntdium hoiulinum toxm selected In a prefeired embodiment the portion ol the toxm compiises the receptor binding domain (i e . the C liagment of the toxm) Th piesent invention is not limited bv the nature ot the non-tox piotem sequence emploved in a preferred embodiment, the non-toxm piotem sequence comprises a polv- histidmc tiact A number ot alternative fusion tags or lusion paitners are known to the art (c g MBP. GST protein A. etc ) and mav be employed lor the pioduction ol lusion proteins compiismg a portion ol a botuhnal toxm The piesent invention further provides a vaccine comprising a tusion protein said lusion protein comprising a non-toxm protein sequence and at least a portion ot a C losliidium hoiulinum toxin said toxin selected from the group consisting ot tvpe B toxm and tvpe I toxin Ihe vaccine mav be a monovalent vaccine (/ c containing onlv a toxin B tusion 5 piotem or a toxin E fusion protein) a bivalent vaccine (/ c containing both a toxin B fusion protein and a toxm E fusion protein) or a trivalent 01 higher vaiencv vaccine In a preferred embodiment the toxin B tusion protein and/oi toxin F tusion protein is combined with a lusion piotem comprising a non-toxm protem sequence and at least a portion ot ( losliidium hoiulinum tvpe A toxm Ihe present invention is not limited bv the nature ot the portion ol
10 the C losliidium hoiulinum toxm selected In a preferred embodiment the portion ot the toxin comprises the leceptor binding domain (/ c the C fiagmeπt ot the toxin) Ihe present invention is not limited bv the natuie ot the non-toxin protein sequence emploved In a pielened embodiment the non-toxin protein sequence compπses a polv-histidme ti act \ number ol alternative lusion tags or lusion partneis are known to the ait (c _j MBP GSI
1 protem \ etc ) and mav be emploved lor the generation ot tusion proteins compπsιn_ι v ccines When a lusion partner (/ c the non-toxin protem sequence) is emploved loi the production ol a iecombinant C botuhnal toxin piotem the fusion pailner mav be lemoved liom the iecombinant ( bolulinal toxin protem ii desned (i t pnoi to administration ol the protem to a subiect) using a vaπetv ot methods known to the ait ( digestion ot tusion
20 pioteins containing 1 actorXa oi thrombin recognition sites with the appropriate enzvtne) \ numbei ot the pi THis vectois emploved herein pro ide an K-terminal his-tag followed bv a (actoiXa cleavage site (see Fxample 28a) the botuhnal C fiamnent sequences follow the I actorXa site and thus [ actorXa can be used to remove the his-tau liom the botuhnal tusion protein In a pieterred embodiment the vaccine is substantiallv endotoxin-liec
->s Ihe present invention is not limited bv the method emploved for the generation of v ccine compiising fusion proteins comprising a non-toxm piotein sequence and at least a poition ol a C losliidium hoiulinum toxin The tusion proteins mav be pioduced bv lecombmant DNA means using either native or svnthetic gene sequences expressed a host cell Ihe piesent invention is not limited to the pioduction ot vaccines using recombinant host cells cell free in
Figure imgf000024_0001
transcπption/tianslation sv stems mav be emploved foi the
11 . expression of the nucleic acid constructs encoding the fusion proteins of the present invention. An example of such a cell-free system is the commercially available TnT™ Coupled Reticulocyte Lysate System ( Promega Corporation. Madison. WI), Alternatively, the fusion proteins of the present invention may be generated by sy nthetic means (i. e.. peptide synthesis).
The present invention further provides a method of generating antibody directed against a Closiridium hoiulinum toxin comprising: a) providing in any order: i ) an antigen comprising a fusion protein comprising a non-toxin protein sequence and at least a portion ol' a Closiridium hoiulinum toxin, said toxin selected from the group consisting of type B toxin and ty pe E toxin, and ii ) a host: and b) immunizing the host w ith the antigen so as to generate an antibody . I n a preferred embodiment, the antigen used to immunize the host also contains a fusion protein comprising a non-toxin protein sequence and at least a portion of" Closiridium hoiulinum ty e A toxin. I he present invention is not limited by the nature of the portion of the Closiridium hoiulinum toxin selected. I n a preferred embodiment, the portion of the toxin comprises the receptor binding domain ( i. e.. the C fragment of the toxin). The present inv ention is not limited by the nature of the non-toxin protein sequence employed. In a preferred embodiment, the non-toxin protein sequence comprises a poly-histidine tract. A number oϊ alternative fusion tags or fusion partners arc know n to the art (e.g.. MBP. GST. protein A. etc. ) and may be employ ed for the generation of fusion proteins comprising v accines. When a fusion partner { i. e.. the non-toxin protein sequence) is employ ed for the production of a recombinant C hoiulinal toxin protein, the fusion partner may be removed from the recombinant O hoiulinal toxin protein if desired ( i. e.. prior to administration of the protein to a subject ) using a variety of methods known to the art { e.g.. digestion of fusion proteins containing FactorXa or thrombin recognition sites with the appropriate enzy me). The present invention is not limited by the nature of the host employed for the production of the antibodies of the invention. In a preferred embodiment, the host is a mammal, preferably a human. The antibodies of the present invention may be generated using non-mammalian hosts such as birds, preferably chickens. In a preferred embodiment the method of the present invention further comprised the step c ) of collecting the antibodies from the host. In yet another embodiment, the method of the present invention further comprises the step d) of purifying the antibodies.
The present invention further provides antibodies raised according to the above methods. The present inv ention further contemplates multivalent vaccines comprising at least two recombinant '. hoiulinum toxin proteins derived from the group consisting of C. hoiulinum serotypes A. B. C I). E. F. and G. Fhe invention contemplates bivalent, trivalent, quadrav alent. pentavaient. heptavalent and septivalent vaccines comprising recombinant ( . hoiulinum toxin proteins. Preferably the recombinant '. hoiulinum toxin protein comprises the receptor binding domain (i.e.. C fragment) of the toxin.
DESCRIPTION OF THE INVENTION fhe present invention contemplates vaccinating humans and other animals with poly peptides derived from C. botulinum neuroloxins which are substantially endotoxin-free. These botuhnal peptides are also useful for the production of antitoxin. Λnti-botulinal toxin antitoxin is useful for the treatment of patients effected by or at risk of symptoms due to the action of ( '. hoiulinum toxins. T he organisms, toxins and individual steps of the present invention are described separately below .
I. Clostridium Species, Clostridial Diseases And Associated Toxins
A preferred embodiment of the method of the present inv ention is directed toward obtaining antibodies against Closiridium species, their toxins, enzymes or other metabolic by products, cell wall components, or synthetic or recombinant versions of any of these compounds. It is contemplated that these antibodies will be produced by immunization of humans or other animals. It is not intended that the present invention be limited to any particular toxin or any species of organism. In one embodiment, toxins from all Closiridium species are contemplated as immunogens. Examples of these toxins include the neuraminidase toxin of ( '. buiyricum. C. sordellii toxins H I and FT. toxins A. B. C I). E. F . and G ol' C hoiulinum and the numerous ( '. perfringens toxins. In one preferred embodiment, toxins A. B and F oi C botulinum are contemplated as immunogens Table 2 above hsts various ( losliidium species, their toxins and some antigens associated with disease
TABLE 2
C lostndial Toxins
Figure imgf000027_0004
It is not intended that antibodies produced against one toxm will oπlv be used against that toxm it is contemplated that antibodies directed against one toxin (t g ( peifnngens tv e A enterotoxin) mav be used as an effective therapeutic against one or more toxιn(s) pioduced bv othei membeis of the genus Closiiidium or other toxin pioducmg oiganisms (c g Bacillus cams
Figure imgf000027_0001
auiais iieptococais muians landobacia LLilcoacciicus Pseudomonas ua
Figure imgf000027_0002
other Pseudomonas species etc ) It is lurthei contemplated that antibodies duected against the portion ot the toxin which binds to mammalian membianes (e g (
Figure imgf000027_0003
enterotoxin A) can also be used against othei oiganisms It is contemplated that these membrane binding domains are pioduced svntheticailv and used as immunouens
II. Obtaining Antibodies In Non-Mammals
A pieleired embodiment ol the method ot the present invention toi obtaining antibodies involves immunization However it is also contemplated that antibodies could be obtained fiom non-mammals without immunization In the case where no immunization is contemplated, the present invention may use non-mammals with preexisting antibodies to toxins as well as non-mammals that have antibodies to whole organisms by virtue of reactions with the administered antigen An example oi the latter involves immunization with synthetic peptides oi recombinant proteins sharing epitopes with whole oigamsm components In a preferred embodiment, the method of the present invention contemplates immunizing non-mammals with bacterial toxιn(s) It is not intended that the piesent invention be limited to any particular toxm In one embodiment, toxm tiom all clostridial bacteria sources (see Table 2) aie contemplated as immunogens I xamples ot these loxms are C huiMiciim neuiammidase toxin, toxins A. B. C D. F. F. and G liom ( botulinum . C perfrtngens toxins a. β. ε. and t. and C sorde/ln toxins HF and I T In a preferred embodiment. C hoiulinum toxins A. B. C 1). E. and F (or fiagments thereot ) aie contemplated as immunogens
\ paiticularly prelerred embodiment involves the use ot baetenal toxin protein oi tiagments ol toxin proteins produced by molecular biological means (i e . iecombinant toxin proteins) In a preteired embodiment, the immunogen comprises the leceptor-bmding domain
(/ e . the 5() kD cai box -terminal poition ot the heavy chain, also lelened to as the C liagment) ol C hoiulinum seiotype A neurotoxin produced bv recombinant F)N \ technology In anothei pieleπed embodiment, the immunogen comprises the leceptoi-bindmg domain ol C hoiulinum serotype B neurotoxin produced by recombinant DN \ technology In vet anothei pretence! embodiment, the immunogen comprises the leceptoi-bmdmg domain legion ol ( hoiulinum serotype F" neurotoxin produced by recombinant DNA teehnologv In vet anothei pieleired embodiment, the immunogen comprises the leceptoi -binding domain icgion ol ( hoiulinum serotype C 1 neurotoxin pioduced by iecombinant DNA technology In yet anothei prelerred embodiment, the immunogen comprises the receptor-binding domain icgion of C botulinum serotype C2 neurotoxin produced by recombinant F)NA technology In vet another preferred embodiment, the immunogen comprises the receptor-binding domain icgion ot C hoiulinum serotype D neurotoxin produced by recombinant DNA technology in vet anothei preferred embodiment, the immunogen comprises the receptor-binding domain icgion ol (' hoiulinum serotype F neurotoxin pioduced bv iecombinant DNΛ technology In yet another pieterred embodiment, the immunogen comprises the leceptor-bmding domain icgion of C hoiulinum seiotype G neurotoxin produced bv iecombinant DNA technology In a preteired embodiment, the recombinant botuhnal toxin pioteins aie expressed as fusion proteins (e ., as histidme-tagged proteins) In a still further prelerred embodiment, the immunogen is a multivalent vaccine comprising the receptoi -binding domain region of C botulinum toxin fiom two or more toxins selected from the group consisting of type A. type B type C (including C 1 and C2). type F). type E. and t pe J toxin hen immunization is used, the preferred non-mammal is liom the class -hrs All birds aie contemplated (e g duck, ostrich, emu. turkev. etc ) A preferred bird is a chicken impoitantiv chicken antibodv does not fix mammalian complement [See H N Benson et al . I Immunol 87616 (1961) ] Thus, chicken ant ody will normally not cause a complement- dependent reaction [A A Benedict and K Yamaga.
Figure imgf000029_0001
and Antihoc Pioduction in Ivian SJK'CICS "in C omparative
Figure imgf000029_0002
(I I Marchalom ed ). pp 335- 375. BlackweTl Oxford (1966) ] Thus the preferred antitoxins ot the present invention will not exhibit complement-ielated side effects observed with antitoxins known piesently hen buds aie used, it is contemplated that the antibodv will be obtained from eithei the bud serum oi the egg \ prelerred embodiment involves collection ol the antibody irom the egg I av mg hens tiansport immunoglobulin to the egg olk ("IgY") in concentrations equal to oi exceeding that found in serum [See R Patterson el al 1 Immunol 89272
(1962) and S B Carroll and B I) Stollar. J Bio) Chem 25824 (1983)] In addition, the Lit c v lume of egg yolk produced vastlv exceeds the volume of serum that can be safelv obtained liom the bird over am given time period Finally, the antibodv horn eggs is purer and moie homogeneous there is far less non-immunoglobuhn protem (as compared to serum) and onlv one class ot immunoglobulin is transported to the volk hen considering immunization with toxins, one mav consider modification ot the toxins to leduce the toxicity In this icgard. it is not intended that the present invention be limned bv immunization with modified toxin Unmodified ("native") toxin is also contemplated as an immunogen It is also not intended that the present invention be limited bv the tvpe oi modification
-- if modification is used The present invention contemplates all tvpes ol toxin modification, including chemical and heat treatment ot the toxin The prefeired modification, however, is loπnaldehv e tieatment
It is not intended that the piesent invention be limited to a particulai mode of immunization, the present invention contemplates all modes ol immunization including subcutaneous, intramuscular, inlrapciitoneal. and intravenous or mtravasculai iiηection as well as pei s administration ot immunogen I he present invention further contemplates immunization with or without adjuvant. ( Ad|uvant is defined as a substance known to increase the immune response to other antigens w hen administered with other antigens.) If ad|uvant is used, it is not intended that the present invention lie limited to any particular type of adjuvant — or that the same adiuvant. once used. be used all the time. While the present invention contemplates all types of ad|iιvant. whether used separately or in combinations, the preferred use of adjuvant is the use of Complete 1 icund' s Adjuvant followed sometime later w ith Incomplete I ιeund"s Adjuvant Anothei preferred use ot adjuvant is the use of Gerbu Adjuvant I he invention also contemplates the use of RIB1 fowl adjuv ant and Quil A adjuvant When immunization is used, the present invention contemplates a wide v aπetv ot immunization schedules. In one embodiment, a chicken is administered toxιn( s) on day zero and subsequently receives toxιn(s) in intervals thereattei It is not intended that the present invention be limited by the particular interv als oi doses Similarly , it is not intended that the present inv ention be limited to any particular schedule loi collecting antibody I he pref ei red collection time is sometime after day 100.
W here birds are used and collection ot antibody is perf ormed by collecting eggs, the eggs may be stoied prior to processing for antibody It is prelerred that eggs be stored at 4°C for less than one y ear
It is contemplated that chicken antibody produced in this manner can be buffei - extiaetcd and used analytically While unpunfied. this preparation can serv e as a reference f or acliv itv ot the antibody prior to further manipulations ( e immunoal tlnity puri fication )
III. Increasing The Effectiveness Of Antibodies
When purification is used, the present inv ention contemplates purif y ing to increase the ef fectiv eness ol both non-mammalian antitoxins and mammalian antitoxins Speciilcallv . the present invention contemplates increasing the percent ot toxin-reactive immunoglobulin I he pref erred purification approach tor avian antibody is poly ethylene gly col ( PEG) sepaiation
Fhe present inv ention contemplates that avian antibody be initially purified using simple, inexpensive procedures In one embodiment, chicken antibody f rom eggs is pun fled by extraction and precipitation with PEG PEG purification exploits the differential solubility ot lipids ( which are abundant in egg yolks) and yolk proteins in high concentrations of PEG 8000 [Poison et al . Immunol. Comm 9 495 ( 1980) J fhe technique is rapid, simple, and relativ ely inexpensive and y ields an immunoglobulin traction that is significantly purer in terms of contaminating non-immunoglobulin proteins than the comparable ammonium sulfate fractions of mammalian sera and horse antibodies. T he majority of the PEG is removed from the precipitated chicken immunoglobulin by treatment with ethanol. Indeed. PEG-purifled antibody is sufficiently pure that the present invention contemplates the use of PEG-purified antitoxins in the passive immunization of intoxicated humans and animals.
IV. Treatment
T he present invention contemplates antitoxin therapy for humans and other animals intoxicated by bacterial toxins. A preferred method of treatment is by intravenous administration of anti-boutlinal antitoxin: oral administration is also contemplated for other clostridial antitoxins.
A. Dosage Of Antitoxin
It w as noted by way of background that a balance must be struck when administering currently available antitoxin w hich is usually produced in large animals such as horses:
.sufficient antitoxin must be administered to neutralize the toxin, but not so much antitoxin as lo increase the risk of untoward side effects. These side effects are caused by: i ) patient sensitivity lo foreign (e.g. horse) proteins: ii) anaphylactic or immunogenic prcφerties of non- immunoglobulin proteins; iii ) the complement fixing properties of mammalian antibodies: and/or iv ) the ov erall burden of foreign protein administered. It is extremely difficult to strike this balance when, as noted above, the degree of intoxication (and hence the level of antitoxin therapy needed ) can only be approximated.
T he present invention contemplates significantly reducing side effects so that this balance is more easily achieved. Treatment according to the present invention contemplates reducing side effects by using PEG-purified antitoxin from birds.
In one embodiment, the treatment of the present invention contemplates the use of PEG-purified antitoxin from birds. T he use of yoik-derived. PFXi-purif ed antibody as antitoxin allows for the administration of: 1 ) non( mammalian)-complemcnt-flxing. avian antibody : 2 ) a less heterogeneous mixture of non-immunoglobulin proteins; and 3 ) less total protein to deliver the equivalent weight of active antibody present in currently available antitoxins. The non-mammalian source of the antitoxin makes it useful for treating patients w ho are sensitive to horse or other mammalian sera. B. Delivery Of Antitoxin
Although it is not intended to limit the route of delivery, the present invention contemplates a method for antitoxin treatment of bacterial intoxication in which delivery of antitoxin is oral. In one embodiment, antitoxin is delivered in a solid form ( e.g.. tablets). In an alternative embodiment antitoxin is delivered in an aqueous solution. When an aqueous solution is used, the solution has sufficient ionic strength to solubilize antibody protein, vet is made palatable for oral administration. The delivery solution may also be buffered (e.g., carbonate buffer pH 9.5) which can neutralize siomach acids and stabilize the antibodies when the antibodies are administered orally. In one embodiment the delivery solution is an aqueous solution. In another embodiment the delivery solution is a nutritional formula. Preferably. the deliv ery solution is infant formula. Yet another embodiment contemplates the delivery of lyophilized antibody encapsulated or microencapsulated inside acid-resistant compounds.
Methods of applying enteric coatings to pharmaceutical compounds are well known to the art | companies specializing in the coating of pharmaceutical compounds are available: for example. Fhe Coating Place ( Verona. WI) and AAI (Wilmington. NC)]. Enteric coatings which are resistant to gastric fluid and whose release (i. e.. dissolution of the coating to release the pharmaceutical compound ) is pFl dependent are commercially available | for example, the polymethacrylatcs F udragit H L and Eudragit® S (Rohm GmbH )]. Eudragit J* S is soluble in intestinal lluid from pl l 7.0; this coating can be used to microencapsulate ly ophilized antitoxin antibodies and the particles are suspended in a solution having a pl l above or below pl l 7.0 for oral administration. The microparticles will remain intact and undissolved until they reached the intestines where the intestinal pH would cause them to dissolve thereby releasing the antitoxin.
The invention contemplates a method of treatment which can be administered for treatment of acute intoxication. In one embodiment, antitoxin is administered orally in either a delivery solution or in tablet form, in therapeutic dosage, to a subject intoxicated by the bacterial toxin which served as immunogen for the antitoxin.
I he invention also contemplates a method of treatment which can be administered proph aelically. In one embodiment, antitoxin is administered orally , in a delivery solution. in therapeutic dosage, to a subject, to prevent intoxication of the subject by the bacterial toxin which serv ed as immunogen for the production of antitoxin. In another embodiment, antitoxin is administered orally in solid form such as tablets or as microencapsulated particles. Microencapsulation oϊ ly ophilized antibody using compounds such as Eudragit® ( Rohm GmbH) or polyethylene glycol . which dissolve at a wide range of pH units, allows the oral administration of solid antitoxin in a liquid form (i c . a suspension) to recipients unable to toleiate administration of tablets (e g . children or patients on feeding tubes) In one preferred embodiment the subject is a child In another embodiment, antibody raised against whole baetenal oi ga sm is administered orally to a subject, in a dehvei y solution, in therapeutic dosage
V . \ accines Against Clostridial Species
I he inv ention contemplates the generation of mono- and multiv alent v accines lot the piotection of an animal (particularly humans) against seveial clostridial species Of particulai interest aie v accines which stimulate the production of a humoral immune response to ( hoiulinum ( i ei am and { diffic ile in humans T he antigens comprising the v accine prepai ation mav be nativ e or recombinantly produced toxin proteins f rom the clostridial species listed above When toxin proteins are used as immunogens thev are generally modified to reduce the toxicity This modification may be by chemical or genetic ( / e . iecombinant DNA technology ) means I n general genetic detoxification ( / e . the expression ol nontoxie ti agments in a host cell ) is preferred as the expression ol nontoxic fragments in a host cell piecludes the piesence of intact, active toxin in the final preparation Howevei . w hen chemical modification is desired the prelerred toxin modification is lormaidehy de treatment
I he inv ention contemplates that recombinant C hoiulinum toxin pi oteins be used as antigens in mono- and multivalent v accine preparations Soluble, substantially endotoxm-free i ecombinant C hoiulinum toxin proteins derived fiom serotypes A. Fϊ and L may be used indiv idually ( / e . as mono-valent v accines) or in combination (/ e . as a multi-valent vaccine) In addition, the recombinant C botulinum toxin proteins derived from serotpes A B and I may be used in conjunction w ith either recombinant or native toxins oi toxoids f i om othei sei oty pes ol ( botulinum. C difficile and C letani as antigens for the pi epai alion of these mono- and multiv alent vaccines It is contemplated that, due to the structural similarity of C hoiulinum and C lelani toxin proteins, a vaccine comprising C difficile and hoiulinum toxin pioteins ( native or i ecombinant or a mixture thereof) be used to stimulate an immune l espouse against C hoiulinum C lelani and ( difficile The present invention further contemplates multi-valent vaccines comprising two or more botulinal toxin proteins selected from the group comprising recombinant C. botulinum toxin proteins derived from serotypes A. B. C (including C l and C2). D. E. F and G. The adverse consequences of exposure to botulinal toxin would be avoided by immunization of subjects at risk of exposure to the toxin with nontoxic preparations which confer immunity such as chemically or genetically detoxified toxin.
Vaccines which confer immunity against one or more of the toxin types A. B. F:,. F and G would be useful as a means of protecting humans from the deleterious effects of those ' hoiulinum toxins known to affect man. Indeed as the possibility exists that humans could be exposed to any of the seven serotypes of C. botulinum toxin (e.g.. during biological warfare or the production of toxin in a laboratory setting), multivalent vaccines capable of conferring immunity against toxin types A-G (including both C l and C2 toxins) would be useful for the protection of humans. Vaccines which confer immunity against one or more of the toxin types C D and E would be useful for veterinary applications. Fhe botulinal neurotoxin is synthesized as a single polypeptide chain which is processed into a heavy ( H: - 100 kD) and a light (L: -50 kD) chain by cleavage w ith proteolvtic enzymes; these two chains are held together via disulfide bonds in the active toxin ( referred to as deriv ative toxin) [ B.R. DasGupta and I F Sugiyama. Biochem. Biophys. Res. C'ommun. 48: 108 ( 1972): reviewed in B.R. DasGupta. J. Physiol. 84:220 ( 1990). H . Sugiyama. Microbiol. Rev. 44:419 ( 1980) and CL. Hatheway. Clin. Microbiol. Rev . 3:66 ( 1990) ]. The heavy chain of the active toxin is cleaved by trypsin to produce two fragments termed 1 1, ( also referred to as H , or C) and 1 1N (also referred to as I E or B). I he H( fragment ( -46 kD) comprises the carboxy end of the 1 1 chain. The H , fragment (-49 kD) comprises the animo end and remains attached to the L chain ( H L). Neither H or HNL is toxic. Hc competes w ith whole derivative toxin for binding to synaptosomes and therefore 1 I( is said to contain the receptor binding site. 'Fhe H( and HN fragments of botulinal toxin are analogous to the fragments C and B of tetanus toxin which are produced by papain cleavage. Fhe C fragment of tetanus toxin has been shown to be responsible for the binding of tetanus toxin to purified gangliosides and neuronal cells ( Halpern and Loftus. J. Biol. Chem. 288: 1 1 1 88 ( 1993)]. Antisera raised against purified preparations of isolated botulinal IT and L chains have been shown to protect mice against the lethal effects of the toxin; however, the effectiveness of the two antisera differ with the anti-H sera being more potent ( I I. Sugiyama. supra). While the different botulinal toxins show structural similarity lo one another, the different serotypes are reported to be immunologically distinct (i e.. sera raised against one toxin type does not cross-react to a significant degree with other types). Thus, the generation of multivalent vaccines may require the use of more than one type of toxin ' hoiulinum toxin genes from all seven serotypes have been cloned and sequenced (Minton (1995). supra); in addition, partial amino acid sequence is available for a number of
C hoiulinum toxins isolated from different strains within a given serotype. The C hoiulinum toxins contain about 1250-1300 amino acid residues. On the DNA level, the overall degree of homology between C botulinum serotypes A, B. C D and E toxins averages between 50 and 60% identity with a greater degree of homology being found between H chain-encoding regions than between those encoding L chains [Whelan el al (1992) Appl Environ.
Microbiol.582345] The degree of identity between C hoiulinum toxins on the amino acid level reflects the level of DNA sequence homology The most divergent area of DNA and ammo acid sequence is found within the carboxy-terminal area of the various (' hoiulinum H chain genes. Fhis portion of the toxin (i e.. H( or the C fragment) plays a ma|oι role in cell binding As toxin from different serotypes is thought to bind to distinct cell receptor molecules, it is not surprising that the toxins diverge significantly over this region
Within a given serotype. small variations in the primary ammo acid sequence ol the botuhnal toxins isolated from different strains has been reported | Whelan el al (1992). supra and Minton (1995). supra]. The present invention contemplates fusion proteins comprising portions ol C hoiulinum toxins from serotypes A-G including the variants found among different strains within a given serotype. The present invention provides oligonucleotide pinners which may be used to amplify the C fragment or receptor-binding region of the toxin gene Irom various strains ol C hoiulinum serotype A. serotype B. serotype C (Cl and C2). serotype 1). serotype E. serotype I and serotype G. A large number of different strains of C hoiulinum serotype A. serotype B. serotype C serotype D serotype E and serotype F are available from the American Type Culture Collection (ATCC; Rockville. MD). For example, the AICC provides the following. Ivpe A strains: 174 (ATCC 3502).457 (ATCC 17862). and NCTC 7272 (ATCC 19397). Type B strains: 34 (ATCC 439).62A (ATCC 7948). NCA 213 B (ATCC 7949). 13114 (ATCC 8083).3137 (ATCC 17780). 1347 (AICC 17841).2017 (ATCC 17843).2217 (ATCC 17844).2254 (ATCC 17845) and VP 1731 (ART 25765).
Type C strains 2220 (ATCC 17782).2239 (ATCC 1778.3).2223 (A FCC 17784; a type C-[\ strain. C'-β stiains produce C2 toxin).662 (ATCC 17849; a type C-α strain; C-α strains produce mainly Cl toxin and a small amount of C2 toxin).2021 (ATCC 17850: a type C-α
JJ strain) and VPI 3803 (ATCC 25766); I ype D strains. ATCC 9633.2023 (ATCC 17851), and VPI 5995 (ATCC 27517); Type L strains. ATCC 43181.36208 (ATCC 9564).2231 (ATCC 17786).2229 (ATCC 17852).2279 (ATCC 17854) and 2285 (ATCC 17855) and Type F strains 202F (ATCC 23387). VPI 4404 (ATCC 25764). VPI 2382 (AICC 27321) and Langeland (ATCC 35415) Type G strain. 113/30 (NCFB 3012) may be obtained from the
National Collection of Tood Bacteria (NCFB. AFRC Institute ol Food Research. Reading, United Kingdom)
Purification methods have been reported tor native toxin types A. B. C D. E. and 1 I reviewed in G Sakaguchi. Pharmac Ther 19165 (1983)] As the diffeient botuhnal toxins aie structurally i elated, the invention contemplates the expression of any of the botuhnal toxins (e g. types A-G) as soluble recombinant fusion proteins
In paiticular. methods tor purification ot the type A botulinum neurotoxin have been developed ]I .) Moberg and H Sugiyama. Appl Environ. Microbiol 35878 (1978)] immunization ol hens with detoxified purified protein results in the generation ot neutralizing antibodies [B S I bailey et al . in Botulinum and lelcinus Nettioioxtns.13 R DasGupta. ed .
Plenum Piess. New York (1993). p 467]
Ihe currently available (' hoiulinum pentavalent vaccine comprising chemically inactivated (/ e . toimaldehyde treated) type A. B. C. D and I toxins is not adequate The efficacy is variable (in particular, only 78% ot recipients produce protective levels ot anti-type B antibodies following administration of the primary series) and immunization is painful
(deep subcutaneous inoculation is required for administration), with adverse reactions being common (moderate to severe local reactions occur in approximately 6% ol recipients upon initial miection. this number rises to approximately I 1% ol individuals who receive booster injections) |Infoιmatιonal Brochure tor the Pentavalent (ABCDI ) Botulinum loxoid. Centers for Disease Control] Preparation ot this vaccine is dangerous as active toxin must be handled by laboratory workers.
In general, chemical detoxification of bacterial toxins using agents such as lormaldehvde. glutaraldehyde or hydrogen peroxide is not optimal for the generation ol vaccines oi antitoxins A delicate balance must be struck between too much and too little chemical modification If the treatment is insufficient, the vaccine may retain icsidual toxicity If the treatment is too excessive, the vaccine may lose potency due to destruction of native mimunogenic determinants Another major limitation ot using botuhnal toxoids for the generation oi antitoxins or vaccines is the high production expense 1 or the above ieasons. the development of methods for the production of nontoxic but immunogenic C botulinum toxin proteins is desirable
The C botulinum and C tetanus toxin proteins have similar structures [reviewed in F I Schantz and E A Johnson. Microbiol Rev 5680 (1992)] The carboxy-terminal 50 kD tiagment of the tetanus toxin heavy chain (fragment C) is released by papain cleavage and has been shown to be non-toxic and immunogenic Recombinant tetanus toxin fragment C has been developed as a candidate vaccine antigen [A J Makoff et al . Bio/Iechnology 71043 ( 1989)| Mice immunized with recombinant tetanus toxin fragment C were protected from challenge with lethal doses of tetanus toxin No studies have demonstrated that the recombinant tetanus fragment C protein confers immunity against other botuhnal toxins such as the C hoiulinum toxins
Recombinant tetanus tiagment C has been expressed in L coli (A I Makoff et al . Bio/ lechnology. sup/a and Nucleic Acids Res 1710191 (1989). J I Halpern et al . Infect Immun 581004 (1990)j. yeast [MA Romanos el al . Nucleic Acids Res 191461 (1991)] and baculovirus [I G Charles et al . Infect Immun 591627 (1991)] Synthetic tetanus toxin genes had to be constructed to facilitate expression in veast (M A Romanos el al . supra) and L coli [Λ I Makoff ei at . Nucleic Acids Res , supui]. due to the high A- f content of the tetanus tox gene sequences High A- 1 content is a common feature ol clostridial genes (M R Popolf ei al . Infect Immun 593673 (1991), H F I aPenotierc et al . in Botulinum and Tetanus B R DasGupta. ed . Plenum Press. New York (1993). p 463] which creates expiession difficulties in coli and yeast due pπmanlv to altered codon usage Itequencv and foituitous polvadenv lation sites, respectivelv
Ihe C tiagment ot the C hoiulinum type A neurotoxin heavy chain has been evaluated as a vaccine candidate Fhe C botulinum type A neurotoxin gene has been cloned and sequenced [D L Thompson el al . Eur J Biochem 18973 (1990)] The C fragment of the type A toxin was expressed as either a fusion protein comprising the botulinal C fragment lused with the maltose binding piotem (MBP) or as a native protein [H F I aPenotiere et al (1993) supia. II 1 LaPenotiere el al . Toxicon 331383 (1995) and Middlebrook and Blown (1995). Cun lop Microbiol Immunol 19589-122] The plasmid construct encoding the native piolein was reported to be unstable, while the fusion protein was expressed primarily in inclusion bodies as insoluble protem Immunization of mice with ciudelv puiified MBP lusion piotem lesulted in protection against IP challenge with 3 LDM1 doses of toxm |t aPenotieie et al . (1993) and (1995). supra] Howevei. this recombinant C hoiulinum type A toxin C fragment/MBP fusion protein is not a suitable immunogen for the production of vaccines as it is expressed as an insoluble protein in E. coli. Furthermore, this recombinant C. botulinum type A toxin C fragment/MBP fusion protein was not shown to be substantially free of endotoxin contamination. Experience with recombinant C. botulinum type A toxin C fragment/MBP fusion proteins shows that the presence of the MBP on the fusion protein greatly complicates the removal of endotoxin from preparations of the recombinant fusion protein (.se Ex. 24. infra). Expression of a synthetic gene encoding C. botulinum type A toxin C fragment as a soluble protein excreted from insect cells has been reported [Middlebrook and Brown ( 1995). supra]: no details regarding the level of expression achieved or the presence of endotoxin or other pyrogens were provided. Like the insoluble protein expressed in E. coli. immunization with the recombinant protein produced in insect cells was reported to protect mice from challenge with C. botulinum toxin A.
Inclusion body protein must be solubihzed prior to purification and/or administration to a host. The harsh treatment of inclusion body protein needed to accomplish this solubilization may reduce the immunogenicity of the purified protein. Ideally, recombinant proteins to be used as vaccines are expressed as soluble proteins at high levels ( i. e.. greater than or equal to about 0.75% of total cellular protein) in E. coli or other host cells (e.g.. yeast, insect cells, etc.). T his facilitates the production and isolation of sufficient quantities of the immunogen in a highly purified form (i. e.. substantially free of endotoxin or other pvrogen contamination). The ability to express recombinant toxin proteins as soluble proteins in E. coli is advantageous due to the low cost of growth compared to insect or mammalian tissue culture cells.
The ( '. hoiulinum type B neurotoxin gene has been cloned and sequenced from two strains oϊ C hoiulinum type B [Whelan el al. ( 1992) Appl. Environ. Microbiol. 58:2345 ( Danish strain) and Hutson et al. ( 1994) Curr. Microbiol. 28: 101 ( Eklund 1 7B strain)]. T he nucleotide sequence of the toxin gene derived from the Eklund 1 7B strain ( ATCC 25765) is available from the EMBL/GenBank sequence data banks under the accession number X71 43: the nucleotide sequence of the coding region is listed in SEQ ID NO:39. The amino acid sequence of the C. botulinum type B neurotoxin derived from the strain Eklund 1 7B is listed in SEQ I D NO:40. The nucleotide sequence of the ( '. botulinum serotype B toxin gene derived from the Danish strain is listed in SEQ ID NO:41 . The amino acid sequence of the C. hoiulinum type B neurotoxin derived from the Danish strain is listed in SEQ ID NO:42.
- jt The C hoiulinum type B neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a di er composed of a light and a heavy chain linked via disulfide bonds The light chain is responsible for pharmacological activitv (/ e . inhibition ol the release ot acetylcholine at the neuromuscular |unctιon) The N-terminal portion ot the heavy chain is thought to mediate channel formation while the C-terminal portion mediates toxm binding, the type B neurotoxin has been reported to exist as a mixture of predominantly single chain with some double chain (Whelan et al supia) T he 50 kD carboxv -terminal portion ot the heav y chain is referred to as the C tiagment or the l lt domain The present invention reports for the first time, the expression of the C fragment of C botulinum tvpe B toxm in heterologous hosts (e g E coli)
The C hoiulinum ty pe T neurotoxin gene has been cloned and sequenced from a numbei ot dif ferent strains [ Poulet el al ( 1992) Biochem Biophys Res Commun 1 83 107 W helan et al ( 1992) 1 ui J Biochem 204 657. and Fujπ el al ( 1993) I Gen Microbiol 1 9 79 | I he nucleotide sequence of the type E toxin gene is available from the EMBL sequence data bank under accession numbers X62089 (strain Beluga) and X62683 (strain NC I C 1 1 2 1 9). the nucleotide sequence ot the coding region (strain Beluga) is listed in SEQ ID NO 45 Hie ammo acid sequence ot the ( hoiulinum ty pe E neurotoxin deriv ed from stiuin Beluga is listed in SEQ I D NO 46 The ty pe F neurotoxin gene is synthesized as a single poly peptide chain which may be converted to a double-chain form (i c . a heav v chain and a light chain) by cleav age w ith trypsm. unlike the ty pe A neurotoxin. the tvpe F euiotoxin exists essentially onlv in the single-chain form The 50 kD cai boxv -terminal poi tion ot the heavy chain is referred to as the C f ragment oi the I I, domain The present inv ention lepoi ts for the first time, the expression of the C fragment ot C hoiulinum type F toxin in heterologous hosts ( e g E coli )
I he ( botulinum ty pe C l u. Y and G neurotoxin genes have been cloned and sequenced The nucleotide and amino acid sequences of these genes and toxins are provided heie I he invention prov ides methods lor the expression of the C fragment from each of these toxin genes in heterologous hosts and the purification of the resulting iecombinant pi oteins
The sub|ect invention provides methods which allow the production ot soluble C holulinuin toxin proteins in economical host cells (e g E coli) In addition the subject inv ention prov ides methods which allow the production ot soluble botuhnal toxin proteins in v east and insect cells I uither methods lor the isolation of purified soluble C hoiulinum
- J toxin proteins which are suitable for immunization of humans and other animals are provided These soluble, purified preparations of C botulinum toxin proteins provide the basis for improved vaccine preparations and facilitate the production of antitoxin
When recombinant clostridial toxin proteins produced in gram-negative bacteria (e g E coli) aie used as vaccines, they are purified to remove endotoxin prior to administration to a host animal In order to vaccinate a host, an lmmunogcnically-effective amount of purified substantially endotoxin-free recombinant clostridial toxin protein is administered in anv ot a numbei ot physiologically acceptable carriers known to the ai t When administered for the purpose ol vaccination, the purified substantially endotoxin-li ee recombinant clostridial toxin protem mav be used alone or in conjunction with known adjutants, including potassium alum, aluminum phosphate, aluminum hydroxide. Gerbu ad|uvant (GmDP. C C Biotech Corp ), R1BI adjuv ant ( MPL, R1BI Immunochemical Research. I no ). QS21 (Cambridge Biotech) The alum and aluminum-based adjutants are particularly preferred when vaccines arc to be administered to humans, however, any adjuvant approved tor use in humans mav be employed I he route ot immunization may be nasal, oral, intramuscular, lntrapeπtoneal or subcutaneous
I he invention contemplates the use ot soluble, substantially endotoxm-f ree preparations ot fusion proteins comprising the C fragment of the ( hoiulinum ty pe A. B. C, D. F. r. and G toxm as vaccines In one embodiment, the v accine comprises the C fragment ot eithei the C hoiulinum type A. B. C D. E. I . or G toxm and a polv-histidme tract (also called a histidine tag). In a particulai ly preferred embodiment, a f usion protem comprising the histidine tagged C fragment is expressed using the pi T senes ol expression vectors ( Nov agen) The pF F expression sy stem utilizes a vector containing the TI promoter which encodes the fusion protein and a host cell which can be induced to express the 1 7 DNA polymerase ( i e . a DE3 host strain) The production of C Iragment fusion proteins containing a histidine tract is not limited to the use ot a particulai expression vector and host strain Seveial commercially available expression vectors and host strains can be used lo express the C fragment protem sequences as a tusion protein containing a histidine tract (I or example, the pQE series (pQE-8. 12, 16. 17. 18. 30, 3 1. 32. 40. 41. 42. 50. 5 1. 52. 60 and 70) ot expression v ectors (Qiagen) which are used with the host strains M I 5fpRE P4] (Qiagen ) and
SG 13009(pREP4 | (Qiagen) can be used to express lusion proteins containing six histidine residues at the amino-terminus of the fusion protem) I urthermore a number ot commercially available expression vectors which provide a histidine tract also provide a protease cleavage site betw een the histidine tract and the protein of interest (e . botulinal toxin sequences) Cleavage of the resulting fusion protein with the appropriate protease will remove the histidine tag from the protein of interest (e g , botulinal toxin sequences) (see Example 28a. infra) Removal of the histidine tag may be desirable prior to administration of the i ecombinant botuhnal toxin protem to a subject (e g . a human)
VI. Detection Of Toxin
Fhe inv ention contemplates detecting bacterial toxin in a sample The term "sample" in the pi esent specification and claims is used in its broadest sense On the one hand it is meant to include a specimen or cultuie On the other hand, it is meant to include both biological and env ironmental samples
Biological samples may be animal, including human, fluid, solid ( . stool ) oi tissue, liquid and solid food products and ingredients such as dairy items, v egetables, meat and meat by -pi oducis. and waste Env ironmental samples include env ironmental material such as sin face matter soil, water and industrial samples, as well as samples obtained Irom food and dairy processing instruments, apparatus, equipment, disposable and non-disposable items I hese examples at e not to be consti ued as limiting the sample ty pes applicable to the present inv ention
I he invention contemplates detecting bacterial toxin by a competitiv e immunoassav method that utilizes recombinant toxin A and toxin B proteins, antibodies raised against i ecombinant bacterial toxin proteins A fixed amount ot the recombinant toxm proteins are immobi l ized to a solid support (c g a microtitcr plate) follow ed bv the addition of a biological sample suspected of containing a bacterial toxin I he biological sample is first mixed w ith al Unity -purified oi PEG fractionated antibodies directed against the recombinant toxm pi otem A l eporter reagent is then added which is capable ot detecting the presence ot antibody bound lo the immobilized toxm protein The reporter substance may comprise an antibody w ith binding specificity tor the antitoxin attached to a molecule which is used to identify the presence of the repoi ter substance II toxin is present in the sample this toxm w ill compete w ith the immobilized recombinant tox protein tor binding to the anti- i ecombinant antibody thereby reducing the signal obtained following the addition ot the rcportei reagent A control is employed where the antibody is not mixed with the sample T his giv es the highest (or reference) signal The invention also contemplates detecting bacterial toxin by a "sandwich" immunoassay method that utilizes antibodies directed against recombinant bacterial toxin proteins. Affinity-purified antibodies directed against recombinant bacterial toxin proteins are immobilized to a solid support (e.g.. microtitcr plates). Biological samples suspected of containing bacterial toxins are then added followed by a washing step to remove substantially all unbound antitoxin, fhe biological sample is next exposed to the reporter substance, which binds to antitoxin and is then washed free of substantially all unbound reporter substance. The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used lo identify the presence of the reporter substance. Identi fication of the reporter substance in the biological tissue indicates the presence of the bacterial toxin.
It is also contemplated that bacterial toxin be detected by pouring liquids (e.g.. soups and other fluid foods and feeds including nutritional supplements for hu ans and other animals ) over immobilized antibody which is directed against the bacterial toxin. I t is contemplated that the immobilized antibody will be present in or on such supports as cartridges, columns, beads, or any other solid support medium. In one embodiment, following the exposure of the liquid to the immobilized antibody, unbound toxin is substantiall remov ed by washing. Fhe exposure of the liquid is then exposed to a reporter substance w hich detects the presence of bound toxin. In a preferred embodiment the reporter substance is an enzyme. Iluorescent dye. or radioactive compound attached lo an antibod w hich is directed against the toxin ( i.e.. in a "sandwich" immunoassay ). I t is also contemplated that the detection sy stem will be developed as necessary (e.g.. the addition of enzyme substrate in enzyme sy stems: observation using Iluorescent light for fluorescent dye sy stems: and quantitation of radioactivity for radioactive systems).
EXPERIMENTAL
T he following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the disclosure which follows, the follow ing abbreviations apply: °C (degrees Centigrade ); rpm ( revolutions per minute): BBS-T Ween (borate buffered saline containing Tvveen ): BSA ( bovine serum albumin); ELISA (enzyme-linked immunosorbent assay ): CFA ( complete Freund's adjuvant ); I FA ( incomplete Freund's adjuvant): IgG ( immunoglobulin G ): IgY ( immunoglobulin Y ): I M (intramuscular); I P ( intraperitoneal ): I V ( intravenous or intravascular); SC (subcutaneous); ILO (water). HC1 (hydrochloric acid), LD|0() (lethal dose lor 100%) of experimental animals), aa (amino acid); HPLC (high performance liquid chromatography ). kD (kilodaltons). gm (grams), μg (micrograms). mg (milligrams), ng (nanogiams). μl (microhters). mi (milhhters); mm (tmlhmeteis). nm (nanometers): μm (miciomeiei ). M (molar). mM (milhmolar). MW (moleeulai weight), sec (seconds), mιn(s)
(minute/minutes). hr(s) (hour/hours). MgCL (magnesium chloride). NaCl (sodium chloride). Na O (sodium carbonate). OI) w (optical density at 280 nm). OD,1(lll (optical density at 600 nm) P \GL (polyacry lamide gel electrophoresis). PBS [phosphate buffeted saline (150 mM NaCT. 10 mM sodium phosphate buffei. pFl 7.2)j. PEG (polyethylene glycol). PMSF (pheny linethy Isulfony 1 fluoride). SDS (sodium dodecyl sulfate). Ins
(tiisthvdioxvmethy Ijaimπoinethane). Lnsure'«' (Fnsuie ι< . Ross Laboratories. Columbus OH). I nlamil K (Fnlamil ι< . Mead Johnson), w/v (weight to volume), v v (volume to volume). \mιeon ( Λmicon Inc . Beveilv MA), \mresco (Λmresco. Ine. Solon. OH). AICC (American Ivpe C ulluie C ollection. Rockville. MD). BBI (Baltimore Biologies I uboratory. (a division ol Becton Dickinson). C ockey sville. MD). Becton Dickinson (F^ecton Dickinson
1 abwaie. I ineoln Paik. \'l). BioRad (BioRad. Richmond. CA). Biotech (C-C Biotech Corp. Powav. C \). Charles River (Charles River Laboratories. Wilmington. MM. C'ocahco (C'ocalico Biologicals Inc . Reamstown. PA). C'ytRx (( \ tRx C rp . Norcross. GA). I aleon (e g Baxtei Healthcare C orp . McCiaw Paik. IE and Becton Dickinson).1 DA (1 ederal I ood and Drug
Figure imgf000043_0001
I ishcr Biotech (Fisher Biotech. Springfield. M. GIBCO (Grand
Island Biologic Company/BRL. Grand Island. NY). Gibco-BRI (I ife leehnologies. Inc Gaitheisbuig. MD). llarlan Sprague Daw ley (Hailan Sprague Daw lev. Ine . Madison. WI). Mullmckio t (a division ol Baxtei Healthcare Corp. McGaw Park. IL). Milhpore (Milhpore C oip . Mailboiough. MA). New England Biolabs (New 1 ngland Biolabs. Inc . Beverly. MA). Novagen (Novagen. Inc.. Madison. W ). Pharmacia (Pharmacia. Inc . Piseatawav. N.I). Qiagen
(Qiagen. C'hatsworth. CΛ). Sasco (Sasco. Omaha. NL). Showdcx (Showa Denko America. Inc . New Yoik. NY). Sigma (Sigma Chemical Co . St I ouis. MO). Sterogene (Sterogene. Ine . C A). lech Lab ( lech Lab. Inc . Blacksburg. VA). and Vaxcell (Va cell. Inc . a subsidiaiy ot CytRX Coip. Norcross. CiA) hen a recombinant piotem is described in the specification it is referred to in a short-hand manner by the ammo acids the toxin sequence piesent in the recombinant prote rounded to the nearest 10 I or example, the recombinant protein pMB 1850-2360 contains amino acids 1852 through 2362 ot the C difficile tox B protein The specification gives detailed construction details for all recombinant proteins such that one skilled in the ail will know precisely which ammo acids are present in a given recombinant protein
EXAMPLE I Production Ot High- liter Antibodies To C losliidium difficil Organisms In A Hen
lo ceita pathogenic oiganisms have been shown to be effective in tieatinu diseases caused bv those organisms It has not been shown whether antibodies can be laised against C losti idium difficile which would be effective in treating infection bv this organism \ccoιdιngly ( difficile was tested as immunogen lor pioduction ot hen antibodies l deteimme the best course loi raising high-titei egg antibodies against whole ( difficile oiganisms. different immunizing strains and different immunizing concentrations were examined Ihe example involved (a) pieparation ol the baetenal immunogen (b) immunization (c) purification ot anti-bacterial chicken antibodies and (d) detection of anti-bactenai antibodies in the purified IgY prepaiations
a) Preparation Ol Bacterial Immunogen
( difficile stiains 43594 (sciogroup \) and 43596 (seiogroup C) were onginullv obtained tiom the AICC I hese two strains were selected because thev lepresent two ot the most commonlv -occurring seiogroups isolated from patients ith antibiotic-associated pseudomembianous colitis (Delmee et al . I Clin Microbiol 28(10)2210 (1990) ] \ddιlιonallv both ol these stiains have been previously characterized with lespcet to then viiulence in the Svπan hamstei model lor ( difficile infection [Delmee a al I Med Miciobiol 3385 (1990) | Ihe bacterial strains were separately cultured on brain heart infusion agar tor 48 hours at 37°C in a Gas Pack 100 lar (BBI Cockeysville MD) equipped with a Gas Pack Plus anaerobic envelope (BBI ) I ortv -eight hour cultures weie used because thev produce bettei growth and the oiganisms have been lound to be moie cross-ieactive with icspect to then surlace antigen presentation Ihe gieatei ihe degree ol cioss-ieaetivitv ol oui lg\ piepaiations the better the probability ot a broad range of activity against different stiains seiogroups |Toma et al I Clin Microbiol 26(3)426(1988) ]
Ihe resulting organisms were removed from the agai suitace using a sterile dacron-tip swab, and weie suspended in a solution containing 04% loimaldehvde in PBS pll 72 I his concentration of formaldehyde has been reported as producing good results for the purpose of preparing whole-organism immunogen suspensions for the generation of polycional anti-C. difficile antisera in rabbits. [Delmee et al.. J. Clin. Microbiol.. 21 :323 ( 1985): Davies et al . Microbial Path.. 9: 141 ( 1990).] In this manner, two separate bacterial suspensions were prepared, one for each strain. The two suspensions were then incubated at 4°C for 1 hour.
Following this period of" formalin-treatment, the suspensions were centrifuged at 4.200 g for 20 min.. and the resulting pellets were washed twice in normal saline. The washed pellets, w hich contained formalin-treated w hole organisms, were resuspended in fresh normal saline such that the v isual turbidity of each suspension corresponded to a Ul McFarland standard. ( M.Λ. Edelstcin. "Processing Clinical Specimens for Anaerobic Bacteria: Isolation and
Identification Procedures." in S.M. Finegold et al (eds.).. Bailey and Scott 's Diagnostic Microbiology, pp. 477-507. C.V. Mosby Co.. ( 1990). The preparation of McFarland nephelometer standards and the corresponding approximate number of oi ganisms for each tube are described in detail at pp. 1 72- 1 73 of this volume. ] Each of the two #7 suspensions w as then split into two separate v olumes. One volume of each suspension was v lumetrically adjusted, by the addition of saline, to correspond to the visual turbidity of a # 1 McFarland standard. ] /</. | The ii ] suspensions contained approximately 3 x 10 organisms/ml. and the "7 suspensions contained approximately 2 x 10'' organisms/ml. [ Id. ] 'Fhe four resulting concentration-adjusted suspensions of formalin-treated C. difficile organisms were considered to be "bacterial immunogen suspensions. " These suspensions w ere used immediately after preparation for the initial immunization. [See section (b). [
T he formalin-treatment procedure did not result in 100% non-viable bacteria in the immunogen suspensions. I n order to increase the level of killing, the formalin concentration and length of treatment were both increased f r subsequent immunogen preparations, as described below in T able 3. (Although viability was decreased with the stronger formalin treatment. 100% inviability of the bacterial immunogen suspensions was not reached. ) Also, in subsequent immunogen preparations, the formalin solutions were prepared in normal saline instead of PBS. At day 49. the day of the fifth immunization, the excess volumes of the four previous bacterial immunogen suspensions were stored frozen at -70°C for use during all subsequent immunizations.
- 4: b) Immunization
1 or the initial immunization.10 ml volumes ot each ot the tour bacterial immunogen suspensions described above were separately emulsified in 12 ml volumes ot CFA (GIBCO) For each ot the loin emulsified immunogen suspensions two loui-month old White Leghorn hens (pre-laving) were immunized (It is not necessary to use pie-laying hens, actively -laving hens can also be utilized ) Each hen received a total volume ot approximatelv 10 ml ot a single emulsified immunogen suspension via lour injections (two subcutaneous and two mtrainuscuiui ) ol approximately 250 μl per site In this mannei a total oi loin diffeient immunization combinations, using two hens per combination were initiated for the purpose ot evaluating both the eflect oi immunizing concentration on egg volk antibodv (IgY) production, and tet train cioss-ieactivity ot IgY taised auainst heterologous stiains The lour immunization groups are summarized in Fable 3
IABLE 3
Immunization Gumps
Figure imgf000046_0001
1 he time point tor the Fust series ol immunizations was designated as "dav zero " All subsequent immunizations weie perlormed as described above except that the bacterial immunogen suspensions vvete emulsified using II A (GIBCO) instead ot C I \ and tor the lalei time point immunization, the stored irozen suspensions weie used instead of tieshlv- piepaied suspensions fhe immunization schedule used is listed in I able 4
TABLE 4
Immunization Schedule
Figure imgf000047_0001
c) Purification Of Λnti- acterial Chicken Antibodies
(iioups ot foui eggs were collected per immunization gioup between davs 80 and 84 post-initial immunization, and chicken immunoglobulin (IgY) was extracted according to a modification ol the procedure ot Λ Poison et al . Immunol Comm . °- 495 (1980) Λ gentle stieam ol distilled water Irom a squirt bottle was used to separate the volks liom the whites, and the volks weie broken bv dropping them through a lunnel into a graduated cvlmdei The lour individual volks were pooled lor each group The pooled, broken volks weie blended with 4 volumes ol egg extraction buffet to improve antibodv v icld (egg extraction buffer is 001 M sodium phosphate.01 M NaCl. pll 75. containing 0005% thimerosal). and PI 0 8000 (Λmiesco) was added to a concentration ol 35% When all the PI.G dissolved, the piotem piecipitates that loimed weie pelleted by centrifugation at 13.000 < g loi 10 minutes Ihe supernatants weie decanted and filtered through cheesecloth lo icmove the lipid layei. and the PI Ci was added to the supernatants to a final concentration ot 12% (the supernatants were assumed to contain 35% PEG) Alter a second centrifugation. the supernatants weie disc ided and the pellets weie centrifuged a final time to extiude the remaining P Cϊ 1 hese crude IgY pellets weie then dissolved in the original volk volume ot egg extraction buffer and stored at 4°C \s an additional control, a preimmune IgY solution was piepared as described above. usmι_ CLMZS collected from u mmunized hens ) Detection Of Anti-Bacterial Antibodies In The Purified IgY
Preparations
In order to evaluate the relative levels of specific anti-C difficile activity in the IgY preparations described above, a modified version of the whole-organism ELISA procedure of N.V. Padhve et al ..1. Clin. Microbiol 29:99-103 (1990) was u.sed. frozen organisms ol both
C difficile strains described above were thawed and diluted to a concentration of approximately 1 x 107 organisms/ml using PBS. pll 7.2. In this way. two separate coating suspensions were prepared, one for each immunizing strain Into the wells ot 96-vvell microtiter plates (falcon. Pro-Bind Assay Plates) were placed 100 μl volumes ol the coating suspensions. In this manner, each plate well received a total ot approximatelv 1 \ 10" organisms ol one strain or the other. The plates were then incubated at 4°C overnight The next morning, the coating suspensions were decanted, and all wells were washed three times using PBS In order to block non-specific binding sites. 100 μl of 05% BSA (Sigma) in PBS was then added to each well, and the plates were incubated foi 2 hours at room temperature Ihe blocking solution was decanted, and 100 μl volumes of the IgY preparations deseiibed above eie initially diluted 1:500 with a solution of 01% BSA in PBS. and then serially diluted in 15 steps fhe following dilutions were placed in the wells. 1.500. 1.2.500. 1.62.5000. 131 .500. and 1 1.562.500. Ihe plates were again incubated for 2 hours at room temperature. I ollowmg this incubation, the IgY-containmg solutions were decanted, and the wells were washed three times using BBS- 1 ween (0.1 M boric acid.0025 M sodium borate.
1.0 M NaCl.01% Tween-20). followed by two washes using PBS-Tween (01%, Iween-20). and mallv. Ivvo washes using PBS only To each well. 100 μl of a 1 7^0 dilution ol rabbit anti-chicken IgG (vvhole-molecule)-alkalιne phosphatase conjugate (Sigma) (diluted in 01% BSA in PBS) was added, fhe plates were again incubated for 2 hours at room temperature. The eoniugale solutions were decanted and the plates were washed as described above, substituting 50 mM Na 'O,. pll 9.5 for the PBS in the final wash fhe plates were developed bv the addition of 100 μl of a solution containing I mg/ml para-nitrophenyl phosphate (Sigma) dissolved in 5() m Na-X'O-. 10 mM MgC pll 95 u> each well, and incubating the plates at room temperature in the dark tor 45 minutes Ihe absorbance ol each well was measured at 410 nm using a Dynatech MR 700 plate reader In this mannei. each ol the four IgY preparations described above was tested for reactivity against both ot the immunizing (' difficile strains: strain-specific, as well as cross-reactive activity was determined fable 5 shows the results of the whole-organism ELISA. All four IgY preparations demonstrated significant levels of activity, to a dilution of 1 62.500 or greater against both of the immunizing organism strains 1 herefore. antibodies raised against one strain were highly cross-i eactiv e w ith the other strain, and vice versa T he immunizing concentration ot oi ganisms did not hav e a significant effect on organism-specific IgY production, as both concenli ations produced approximatelv equivalent responses Therefore, the lower immunizing concenti ation ot approximatelv I 5 x 10s organisms/hen is the preferred immunizing concentration of the two tested I he preimmune IgY preparation appeared to possess relativ ely low lev els of ( " c/////c ;/e-reactιv e activity to a dilution of 1 .500. probably due to pπoi exposure ol the animals to environmental clostridia.
\n initial w hole-organism f LISA was perlormed using IgY preparations made from single C I) 43594. tt \ and CD 43596. ≠ \ eggs collected around dav 50 (data not shown ) Specific uici s wei e found to be 5 to 1 -fold lower than those leported in Table 5 These l esults demonstrate that it is possible to begin immunizing hens prior to the time that thev begin to lav eggs, and to obtain high titer specific IgY irom the first eggs that are laid In othei words, it is not necessary to wait foi the hens to begin lav ing betore the immunization schedule is slai ted
TABLE 5 Results Of The Aιιtι-C difficile Whole-Oiga sm ELISA
Figure imgf000050_0001
EXAMPLE 2
Treatment Of ( ' difficile Infection With Antι-( difficile Antibodv
In order to determine whether the immune IgY antibodies raised against whole ( difficile organisms were capable of inhibiting the infection ol hamsters bv (' difficile. hamsters infected bv these bacteria were utilized. [1 yerlv et al . Inteet Immun..592215- 2218 (1991). I Phis example involved, (a) determination of the lethal dose ol (' difficile organisms: and (b) treatment oϊ infected animals with immune antibodv or control antibodv in nutritional solution a) Determination Of The Lethal Dose Of C. difficile Organisms
Determination ot the lethal dose of C difficile organisms was carried out according to the model described bv D M Lyeilv a al Infect Immun .592215-2218 ( 1991 ) ( difficile stiam ICC 43596 (serogroup C ATCC) was plated on BHI agar and grown anaerobicallv (BBI Gas Pak 100 system) at 37°C for 42 houis Organisms were removed from the agar suitace using a sterile ducron-tip s ab and suspended in stenle 09% NaC 1 solution to a densnv ot 10s oiga sms/ml
In order to determine the lethal dose ol C difficile in the presence ol control antibodv and nutritional formula, non-immune eggs were obtained fiom unimmunized hens and a 12% P G piepaiation made as described in Example 1(c) This preparation was redissolved in one lourth the onginul volk volume ol vanilla flavor Ensure"
Starting on
Figure imgf000051_0001
one. groups ot female Golden Syrian hamsters (llarlan Spiague Dawlev) 89 weeks old and weighing approximatelv 100 gm weie oiallv admmisteied 1 ml ol the pieimmune 1 nsuien formula at tune zeio.2 houis 6 houis and 10 houis \t 1 houi. animals were oiallv admmisteied 30 mg clmdamvcin HCI (Sigma) in I ml of atei This ug predisposes hamsteis to ( difficile infection bv altering the normal intestinal flora On dav two the animals were given 1 ml ot the preimmune lgY/Ensure« formula at time zero.2 hours 6 hours and 10 hours At 1 hour on dav two different groups ot animals were inoculated oiallv with saline (control) oi 10\ \(Ϋ 10" oi 10* L difficile oiganisms in I ml of saline I rom davs 3-12 animals were given I ml of the preimmune lg /I nsuιe'< foimula ihiee times daily and observed tor the onset of diarrhea and death L aeh animal was housed in individual cage and was ottered lood and watei ail libitum ol 10' - 10s oiganisms resulted in death in 3-4 davs while the lower doses ol 10 - I 04 organisms caused death in davs Cecal swabs taken liom dead animals indicated the presence ol C difficile Given the effectiveness ot the 1 ' dose, this number of organisms was chosen for the following experiment to see it hypeπmmune unti-t difficile antibodv could block infection
b) I rcatment Of Infected Animals With Immune Antibody Or Control Antibodv In [Nutritional Formula
Ihe cxpeiiment in (a) was lepeated using three groups ol seven hamsteis each Gioup A icceived no chndumvcin or difficile and was the survival contiol Gioup B received chndamvem. I(V ( difficile organisms and preimmune IgY on the same schedule as the animals in (a) above. Group C received clindamycin. 10" C difficile organisms, and hyperimmune anti-C difficile IgY on the same schedule as Group B. The anti-C. difficile IgY was prepared as described in E.xample 1 except that the 12%) PEG preparation w as dissolved in one fourth the original yolk volume of Ensured.
All animals were observed for the onset of diarrhea or other disease symptoms and death. Each animal was housed in an individual cage and was offered food and water ad libitum. The results are shown in Table 6.
TABLE 6
The Effect Ol" Oral Feeding Of Hyperimmune IgY Antibody on ( '. difficile Infection
Figure imgf000052_0001
Mean of seven animals.
Hamsters in the control group A did not develop diarrhea and remained healthy during the experimental period. Hamsters in groups B and C developed diarrheal disease. Anti-C difficile IgY did not protect the animals from diarrhea or death, all animals succumbed in the same time interval as the animals treated with preimmune IgY. Thus, w hile immunization with w hole organisms apparently' can improve sub-lethal sy mptoms with particular bacteria (see U.S. Patent No. 5.080.895 to I I. Tokoro). such an approach does not prov e to be productive to protect against the lethal effects of C. difficile.
EXAMPLE 3
Production of ( '. hoiulinum Type A Antitoxin in Hens
In order to determine w hether antibodies could be raised against the toxin produced b clostridial pathogens, w hich would be effective in treating clostridial diseases, antitoxin to ( '. hoiulinum type A toxin was produced. This example involves: ( a) toxin modi fication: ( b) immunization: ( e ) antitoxin collection: ( d) antigenicity assessment: and ( e ) assav of antitoxin titer. a) Toxin Modification
( hoiulinum tvpe A toxoid was obtained from B R DasGupta From this, the active type \ neurotoxin (M W approximatelv 150 kD) was purified to greater than 99% puntv according lo published methods [B R DasGupta & V Suthvamooithv Toxicon 22415 5 (1984) ] Ihe neurotoxin was detoxified with formaldehyde accoidmg to published methods
| R Singh &. B R DasGupta loxicon 27403 (1989) |
b) Immunization
( hoiulinum toxoid lor immunization was dissolved in PBS (1 mg/ml) and was 10 emulsified with an approximatelv equal volume of C A (GIBCO) toi initial immunization or
II \ toi boostei immunization On dav zeio two white lcghoin hens obtained tiom local bieedeis weie each injected at multiple sites (intramuscular and subcutaneous) with 1 ml inactivated toxoid emulsified in 1 ml f \ Subsequent boosici immunizations were made accoidin to the following schedule lor dav of injection and toxoid amount davs 14 and 21 - I 05 nig άa\ 171 - 075 mg davs 394 401 409 - 025 mg One hen received an additional boostei ol 015() mg on dav 544
Figure imgf000053_0001
C ollcction
1 otal volk immunoglobulin (IgY) was extracted as described in Example 1(e) and the 0 IL pellet was dissolved in the onginal volk volume ol PBS ith thimeiosa!
d) \ntιgcnιeιtv Assessment
1 g<_s were collected from das 409 through dav 423 to assess whether the toxoid was sufficiently immunogenic lo laise antibodv L-ggs tiom the two hens weie pooled and
2^ antibodv was collected as desenbed m ihe standard PI G protocol [Example 1(c) ]
\ntιgcmeιtv ot the botuhnal tox was assessed on Western blots The 150 kD detoxified tvpe \ neurotoxin and unmodified toxic 300 kD botuhnal tvpe A complex (toxin used toi mtragastric route administration lot animal gut neutralization experiments see I xumple 6) were separated on a SDS-poivacrv lamide reducing gel Ihe Western blot technique was
30 pei toi med according to the method of rovvbin [II lowbin <_/<:// Pioc Natl \cad Sci
1 SΛ 764350 (1979) | I en μg samples ot ( hoiulinum complex and toxoid weie dissolved in SDS i educing sample buffer (1% SDS 05% 2-mercaptoethanol 50 mM Ins pll 68 10% glvcerol 0025% w/v bromphenol blue 1 % β-mercaptoethanol) heated at 95°C for 10 min
- s\ and separated on a 1 mm thick 5% SDS-polyacrylamide gel [K.. Weber and M. Osborn."/Vo/t'/ and Sodium Dodecvl Sulfate Molecular Weight Determination on Polvacrylamide dels and Related Piocedures " in The Proteins.3d Edition (H Neurath & R L Hill. eds). pp. 179-223, (Academic Press. NY.1975) ] Pait of the gel was cut off and the proteins were stained with Coomassie Blue. Ihe proteins in the remainder of the gel weie transferred to nitrocellulose using the Milhbiot-SDE electio-blotting system (Milhpore) according to manufacturer's directions The nitrocellulose was temporarily stained with 10% Ponceau S [S.B. Carroll and A. Eaughon. "Production and Purification of Polycional Antibodies to ihe foreign Segment of β-galaclosidase fusion Proteins " in DK 1 Cloning t Piaciical i/ψroach. Vol. III. (D Glover, ed.). pp 89-111. IR1 Press. Oxford. (1987)] to visualize the lanes, then destamed bv running a gentle stream ol distilled water over the blot loi several minutes Ihe nitrocellulose was immersed in PBS containing 3% BSA overnight at 4°( to block anv remaining protein binding sites fhe blot was cut into snips and each strip was incubated with the appropriate primary antibodv Ihe avian anti-C botulinum antibodies [described in (c)[ and pre-immune chicken antibodv (as control) weie diluted 1 125 in PBS containing 1 mg/ml BS \ lor 2 hours at room temperature Ihe blots weie washed with two changes each ol laige volumes ol PBS. BBS- Iween and PBS. successively (10 m in/wash) Goat anti-chicken IgG alkaline phosphatase coniugated secondary antibody (1 isher Biotech) was diluted 1500 in PBS containing 1 mg/ml BSA and incubated with the blot tor 2 hours at ioom temperature Ihe blots weie washed with two changes each of laige volumes ot PBS and BBS- 1 ween, lollowed bv one change ot PBS and 01 M Tπs-lICI. pll 95 Blots were developed in lreshlv prepared alkaline phosphatase substrate butlei (100 μg/ml nitroblue tetrazohum (Sigma).50 μg'ml 5-bιomo-4- chloro-3-mdolv phosphate (Sigma).5 mM MgCU in 50 mM Na-,C(),. pll 95) Ihe Western blots are shown in 1 igure 1 Ihe anli-C hoiulinum IgY reacted to the toxoid to give a broad immunoreactive band at about 145-150 kD on the reducing gel I his toxoid is relractive to disulfide cleavage by reducing agents due to lormalm ciosslmkmg Ihe immune IgY reacted with the active toxin complex, a 97 kD ( hoiulinum ivpe A heavy chain and a 53 kD light chain The preimmune IgY was unreactive to the ( hoiulinum complex oi toxoid m the Western blot c) Antitoxin Antibodv Titer
The IgY antibodv titer to C botulinum tvpe A toxoid of eggs harvested between dav 409 and 423 was evaluated bv I LISA prepared as follows Ninetv-six-well falcon Pro-bind plates eie coated overnight at 4°C with 100 μl/well toxoid [B R Singh &. B R Das Gupta - loxicon 27403 (1989)| at 25 μg/ml in PBS pH 75 containing 0005% thimerosal Ihe following dav the wells weie blocked with PBS containing 1% BSA lor 1 hour at 37°C he IgY liom immune or preimmune eggs was diluted in PBS containing 1% BSA and 005% 1 ween 20 and the plates were incubated tor 1 hour at 37°C Ihe plates were washed three limes with PBS containing 005% [ween 20 and three times with PBS alone Mkaline
10 phosphatusc-coi ugated goat-anti-chicken IgG (Fisher Biotech) was diluted 1750 in PBS containing 1% BSA and 005% 1 ween 20 added lo the plates and incubated 1 hour at 37°C Ihe plates weie washed as betoie and p-mtrophenv I phosphate (Sigma) at 1 mg/ml in 005 M \a CO pll 9 s |() m\1 MgC was added
1 he tesults aie shown in I igure 2 ( hickens immunized with the toxoid generated
I "> high liteis ol antibodv to the immunogen Importantly eggs liom both immunized hens had significant anti-unmunogen antibodv liteis as compared to preimmune eonltol eggs Ihe anti- ( hoiulinum Ig^ possessed significant activity to a dilution ol 193750 or greutei
EXAMPLE 4 0 Prepaiation Ot Avian Egg Yolk Immunoglobulin In An Orally Λdministrable ioim
In ordei to admmistei avian IgY antibodies oiallv to expeinπeπtal mice an effective delivery foimula lor the IgY had to be determined The concern was that it the etude lg\ was dissolved in PBS the salme in PBS would dehvdrate the mice which might prove
2^ harmful over the duialion ol the studv Therefore alternative methods ol oial administration ot IgY weie tested The example involved (a) isola-tion ot immune IgY (b) solubihzation ol IgY in watei oi PBS including subsequent dialysis ot the IgY-PBS solution with watei to eliminate oi i educe the salts (salt and phosphate) in the butfei and (e) comparison ot the quantity and activity of leeovered IgY bv absoibance at 280 nm and PΛG1 and enzvme-
30 linked immunoassay (ELISA)
- 5 i a) Isolation Of Immune IgY
In ordei to investigate the most effective delivery formula for IgY. e used IgY which was raised against ( loialus dunssus lerrificus venom Three eggs were collected fiom hens immunized with the C dunssus teiiificus venom and IgY was extracted tiom the yolks using the modified Poison piocedure described bv Thallev and ( anoll |Bιo/Iechnologv .8934-938
(1990)] as described in Example 1(c)
The egg yolks were separated trom the whites, pooled and blended with four volumes ot PBS Powdered PEG 8000 was added to a concentiation of 35% fhe mixture was eentiifuged at 10.000 ipm toi 10 minutes to pellet the pi capitated protein, and the supernatant was filtered thiough cheesecloth to remove the lipid lavei Powdered PI G 8000 was added to the supernatant to bung the final PFG concentration to 12% (assuming a PEG concentration ol 35% in the supernatant) Ihe 12% PEG/IgY' mixtute was divided into two equal volumes and centrifuged to pellet the IgY
) Solubilization Of The IgY In Water Or PBS
One pellet was resuspended in 12 the original volk volume ol PBS. and the othei pellet was icsuspended in 1/2 the onginul volk volume ol watei Ihe pellets weie then centrifuged to remove anv paitieles oi msoluble material Ihe \<.\ in PBS solution dissolved icadilv but the traction icsuspended in water remained cloudv In oidei to satisfy anticipated sterility requiicments toi oiallv admmisteied antibodies the antibodv solution needs to be llllei -sterilized (as an alternative lo heat sterilization which would desliov the antibodies) Ihe preparation ol IgY icsuspended in water was too cloudv to pass thiough either a 02 oi 045 μm membrane filter so 10 ml ol the PBS resuspended liaction was dial} zed overnight al loom temperature against 250 ml ot water Ihe following morning the dialvsis chamber was emptied and refilled with 250 ml ol liesh II O toi a second dialvsis Thereafter, the yields ol soluble antibodv were determined at OD „„ and are compaied in 1 able 7 TABLE 7
Dependence Of IgY Yield On Solvents
Figure imgf000057_0001
Resuspendmg the pellets in PBS followed bv dialysis against water recovered more antibodv than directly resuspending the pellets in water (77% versus 61 %). Equivalent v olumes ot the IgY' preparation in PBS or water were compared by PAGE., and these results were in accordance w ith the absorbance values (data not shown)
e) Activity Of IgY Prepared With Different Solvents
An 1 1 ISA was perlormed to compare the binding activ ity ol the IgY extracted by each procedure described abov e ( ' dunssus lerrificus (C d t ) v enom at 2 5 μg/ml in PBS w as used to eoat each well tit a 96-well mrcrotiter plate. I he remaining protem binding sites were blocked w ith PBS containing 5 mg/ml BSA Primary antibodv dilutions ( in PBS containing I mg'ml BSA ) w ere added in duplicate After 2 hours ol incubation at room temperature, the unbound pπmarv antibodies were removed bv washing the wells w ith PBS. BBS-Tween. and PBS I he species specific secondary antibodv (goat anti-chicken immunoglobulin alkalme-phosphatase conjugate (Sigma) was diluted 1 .750 in PBS containing I mg/ml BS A and added to each wel l of the microtiter plate After 2 hours of incubation at l oom temperature, the unbound secondary antibody was removed by w ashing the plate as betore. and freshly prepared alkaline phosphatase substrate ( Sigma) al 1 mg/ml in 50 mM NaX'O-. 10 mM MgCE. pl l 9.5 was added to each well, fhe color development was measured on a Dynatech MR 700 microplatc reader using a 412 nm filter f he results are show n in fable 8.
I he binding assay results parallel the recovery values in Table 7. w ith PBS-dissolved IgY' show ing slightly more activ ity than the PBS-dissolved/I EO dialyzed antibodv . fhe water-dissolved" antibodv had considerably less binding activ ity than the other preparations. EXAMPLE 5
Survival Of Antibodv Activitv After Passaue Throuuh The Gastrointestinal Tract
In order to determine the feasibility ot oral administration of antibody, it was ot interest to determine whether orally administered IgY survived passage through the gastrointestinal tract The example involved, (a) oral administration ot specific immune antibodv mixed with a nutritional formula, and (b) assav ot antibodv activity extracted from leces
TABLE 8
Antigen-Binding Activity Ol IgY Prepared With Dillerent Solvents
Figure imgf000058_0001
a) Oral Administration Of Antibody
Ihe IgY' preparations used in this example are the same PBS-dιssolved/I EC) dialvzed antivenom materials obtained in Example 4 above, mixed with an equal volume of Enfamιfκ I wo mice were used in this experiment, each receiving a different diet as lollows
1 ) w ter and food as usual.
2) immune IgY preparation dialyzed against water and mi.xed I I with Enfamil'R
( Ihe mice were given the corresponding mixture as their onlv source ot lood and water)
b) Antibody Activity After Ingestion
Alter both mice had ingested their respective tluids. each tube was refilled with approximately 10 ml of the appropriate fluid first thing in the moin g Bv mid-mornmg there was about 4 lo 5 ml of liquid left in each lube At this point stool samples were collected tiom each mouse, weighed, and dissolved in approximately 500 μl PBS per 100 mg stool sample. One hundred and sixty mg of control stools (no antibodv) and 99 mg of experimental stools (specific antibody) in 15 ml microfuge tubes were dissolved in 800 and 500 μl PBS. respectively. Ihe samples were heated at 37°C foi 10 minutes and vortexed vigorously fhe experimental stools were also broken up with a narrow spatula Each sample was centrifuged for 5 minutes in a microfuge and the supernatants. presumably containing the antibodv extracts, were collected The pellets were saved at 2-8°C in case future extracts were needed Because the supernatants were tinted, they were diluted five-fold in PBS containing 1 mg/ml BSA for the initial dilution in the enzyme immunoassay ( ELISA) The primary extracts were then diluted five-fold serially from this initial dilution. I he volume of primary extract added to each well was 190 μl. The ELISA was performed exactly as described m Example 4.
TABLE 9
Specific Antibodv Activitv Alter Passage t hrough The Oasiiomtesunal I raci
Figure imgf000059_0001
I here was some active antibodv in the fecal extract fi om the mouse giv en the specific antibodv in 1 ntamιl κ formula, but it was present at a v ery low lev el. Since the samples were assav ed at an initial 1 .5 dilution, the binding observed could have been higher w ith less dilute samples ( onseqiienth . the mice were allowed to continue ingesting either regular food and water oi the specific IgY' in EnfamiHi formula, as appropriate, so the assay could be repeated Another I 1 ISA plate was coated ov ernight with 5 μg/ml of C el t v enom m PBS
I he lol low mg morning the 1 LISA plate w as blocked w ith 5 mg/ml BSA. and the lecal samples w ere extracted as before, except that instead of heating the extracts at 70C. the samples w ei e kept on ice lo limit proteolv sis The samples were assayed undiluted initially , and in 5X serial dilutions thereafter Otherwise the assav was carried out as before
TABLE 10
Specific Antibodv' Survives Passage Through The Gastrointestinal Tract
Figure imgf000060_0001
The experiment confirmed the previous results, with the antibody activity markedly- higher, fhe control fecal extract showed no anti-C. /. /. activity, even undiluted, while the fecal extract from the anti-( '. d. t. IgY/Enfarnth&-fcd mouse showed considerable anti-C c/. /. activity. This experiment (and the previous experiment ) clearly demonstrate that active IgY antibodv survives passage through the mouse digestiv e tract, a finding w ith favorable implications for the success of IgY antibodies administered orally as a therapeutic or prophylactic.
EXAMPLE 6
In Vivo Neutralization Of Type ( ' hoiulinum Tv pe A Neurotoxin By Avian Antitoxin Antibodv
This example demonstrated the ability of PEG-purified antitoxin, collected as described in Example 3. to neutralize the lethal effect of ( '. hoiulinum neurotoxin type A m mice. To determine the oral lethal dose ( ED1 ()) of toxin A. groups of BALB/c mice w ere given different doses of toxin per unit body weight (average body weight of 24 grams), for oral administration, toxin A complex, which contains the neurotoxin associated w ith other non-toxin proteins was used. This complex is markedly more toxic than purified neurotoxin when given by the oral route. | I . Ohishi et al.. Infect. Immun.. 16: 106 ( 1977). ] ( '. hoiulinum toxin type A complex, obtained from Eric Johnson ( University' Oϊ Wisconsin. Madison ) was 250 μg/ml in 50 mM sodium citrate, pl l 5.5. specific toxicity 3 1 0 ' mouse ED„,/mg w ith parenterai administration. Approximately 40-50 ng/gm body weight was usuallv fatal w ithin 48 hours in mice maintained on conventional food and water. When mice were given a diet ad libitum of only EnfamilW the concentration needed to produce lethality was approximately 25 times higher (125 ng/gm bodv weight) Botuhnal toxm concentrations ot approximately 200 nggm body weight were fatal in mice led Enfamil® containing preimmune IgY (resuspended in Lnfamilw at the original volk volume)
The oral LD,(H1 ol ( hoiulinum toxin was also determined in mice that received known amounts of a mixture of pteimmune IgY-Lnsure<ι<) delivered oiallv through feeding needles Using a 22 gauge feeding needle mice were given 250 μl each of a preimmune IgY -f nsuie ι< mixture (preimmune IgY dissolved in 1/4 original volk volume) I hour befoie and I 2 hour and 5 houis alter administering botuhnal tox loxin concentrations given orallv langcd Irom approximately 12 to 312 ng/gm bodv weight (03 to 75 μg per mouse) Botuhnal tox complex concentration of approximately 40 ng/gm bodv weight (1 μg per mouse) was lethal in all mice in less than 36 hours
I wo gioups of BALB/c mice 10 per group were each given orallv a single dose of 1 ug each ol botuhnal tox complex in 100 μl ot 50 mM sodium citiatc pH 55 [he mice leceived 250 μl ticatments ot a mixture ot cither preimmune oi immune IgY m I nsuien (14 onginul volk volume) I hour betore and 1 '1 hour 4 hours and 8 hours alter botulinal toxm admmistiation The mice received three treatments pei dav toi two moie davs fhe mice were observed lor 96 hours Ihe survival and mortality are shown in [able 11
TABLF II
Neutralization Of Botu nal fovin A In I >
Figure imgf000061_0001
\ll mice tieated with the pieimmune lgY-rnsuιei< mixtuie died within 46 houis post- loxm admmistiation Ihe average time ot death in the mice was 32 bouts post toxin udministiution lieatments of preimmune IgY-Lnsure^ mixture did not continue bevond 24 houis due to extensive paralvsis ot the mouth in mice of this group In contrast all ten mice tieated with the immune anti-botuhnal toxin IgY -Ensured mixtuie sur ived past 96 houis Only 4 mice in this group exhibited svmptoms of botulism toxiutv (two mice about 2 davs aftei and two mice 4 davs after toxin administration) These mice eventually died 5 and 6 davs later Six of the mice in this immune group displayed no adveise effects to the toxin and remained alive and healthy long term Thus the avian anti-botuhnal toxin antibodv demonstialed veiv good protection liom the lethal effects of ihe toxm in the experimental EXAMPLE 7
Production Of An Avian Antitoxin Against Closiridium difficile Toxin A
I'oxin A is a potent cytotoxin secreted by pathogenic strains of C difficile, that plays a direct role damaging gastrointestinal tissues In more severe cases of (' difficile intoxication, pseudomembranous colitis can develop which may be fatal f his wouid be prevented bv neutralizing the effects of this toxm in the gastrointestinal tract As a first step, antibodies were produced against a portion of the toxm The example involved (a) con|ugatιon ol a synthetic peptide of toxin A to bovine serum albumin: (b) immunization oϊ hens with the peptide-BSA conjugate; and (c) detection ol antitoxin peptide antibodies bv
LLISA
a) Conjugation Of A Synthetic Peptide Of Toxin A To Bovine
Serum Albumin Ihe synthetic peptide CQ flDGKKYYJ'N-NIE (SEQ ID NO 82) was prepared commercially (Multiple Peptide Systems. San Diego. CA) and validated lo be 80% pure bv high-pressuie liquid chromatograph} The eleven amino acids lollowmg the evsteine icsidue represent a consensus sequence of a repeated amino acid sequence lound l xin \ [Wren cl al . Infect Immun..59.3151-3155 (1991) | The evsteine was added to facilitate conjugation to carrier protein
In older to prepare the carrier for conjugation. BSA (Sigma) was dissolved in 001 M NaPO,. pll 70 to a final concentration of 20 mg/ml and n-maleimidobenzoyl-N- hvdroxv succmimide ester (MBS. Pierce) was dissolved in N.N-dnnethvT lo namide lo a concentration ot 5 mg/ml MBS solution.051 ml. was added to 325 ml oi the BSA solution and incubated foi 30 minutes at room temperature with stirring every 5 minutes The MBS- activated BSA was then purified by chromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 M NaPO,. pll 7.0 buffer Peak i actions were pooled (60 ml)
I vophihzed toxin A peptide (20 mg) was added to the activated BSA mixture, stirred until the peptide dissolved and incubated 3 hours at loom temperatuie Within 20 minutes, the reaction mixture became cloudy and precipitates formed Alter 3 hours, the reaction mixture was centrifuged at 10.000 x g for 10 mm and the supernatant analyzed for protem content. No significant protein could be detected at 280 nm 1 he conjugate precipitate was washed three times with PBS and stored at 4°C. A second conjugation was performed with 15 mg of activated BSA and 5 mg of peptide and the conjugates pooled and suspended at a peptide concentration of 10 mg/ml in 10 mM NaP04. pH 7.2.
b) Immunization Of Hens With Peptide Conjugate
Two hens were each initially immunized on day zero by injection into two subcutaneous and two intramuscular sites with 1 mg of peptide conjugate that was emulsified in CTA ( GI BCO). The hens were boosted on day 14 and day 21 with 1 mg of peptide conjugate emulsified in I EA ( GIBCO).
e) Detection Of Antitoxin Peptide Antibodies By ELISA
IgY' was purified from two eggs obtained before immunization ( pre-immune) and two eggs obtained 3 1 and 32 days after the initial immunization using PEG fractionation as described in Example 1 .
Wells of a 96-well mierotiter plate ( falcon Pro-Bind Assay Plate) were coated overnight at 4°C with 100 μg/ml solution of the toxin A synthetic peptide in PBS. pH 7.2 prepared by dissolving 1 mg of the peptide in 1 .0 ml of H ,0 and dilution oϊ PBS. The pre- immune and immune IgY preparations were diluted in a live-fold series in a buffer containing 1 % PEG 8000 and 0. 1 % Tween-20 (v/v) in PBS. pl l 7.2. The wells were blocked for 2 hours at room temperature with 150 μl of a solution containing 5% ( v/v) Carnation* nonfat d v- milk and 1 % PEG 8000 in PBS. pl l 7.2. After incubation for 2 hours at room temperature, the wells were washed, secondary rabbit anti-chicken IgG-alkaline phosphatase ( 1 :750 ) added, the wells washed again and the color development obtained as described in E.xample 1 . The results are shown in Table 12.
TABLE 12
Reactivity Of IgY With Toxin Peptide
Figure imgf000063_0001
Clearly, the immune antibodies contain liters against this repeated epitope of toxin A. EXAMPLE 8
Production Of Avian Antitoxins Against (losliidium difficile Native loxins A And B
lo determine whether avian antibodies are effective tor the neutralization ot (' difficile toxins, hens were immunized using native ( difficile toxins A and B The resultnm egg volk antibodies were then extracted and assessed for their ability to neutralize toxins A and B in vitro The Example involved (a) preparation ol the toxin immunogens. (b) immunization, (e) purification of the antitoxins, and (d) assay ot toxin neutralization activity
a) Preparation Of The Toxin Immunogens
Both difficile native toxins A and B. and C difficile toxoids. piepaied b} the tieatment ol the native toxins with tormaldehvde. were emploved as immunogens ( difficile toxoids A and B were prepared bv a procedure which was modified from published methods (Ehπch el al . Inteet Immun 281041 (1980) Separate solutions (in PBS) of native ( difficile toxin A and toxin B (Tech Lab) were each ud|usted to a eoncentiation ot 020 mg/ml. and tormaldehvde was added to a final concentration ot 04% Ihe toxin/tormaldehvde solutions were then incubated at 37°C lor 40 hrs I ree loimaldehvde was then removed Irom the resulting toxoid solutions by dialvsis against PBS at 4°( In pieviouslv published lepoits this dialvsis step was not performed Therefore, tree tormaldehvde must have been present then toxoid pieparations Ihe toxoid solutions were concentrated, using a entnprep eoneentratoi unil (Amicon). to a
Figure imgf000064_0001
toxoid concentration ol 40 mg/ml Ihe two lesulting prepai lions were designated as toxoid A and toxoid B
( difficile native toxins were piepared b} concentrating stock solutions ot toxm A and toxin B ( lech I ab. Inc). using C entπprep concentrator units (Amicon). to a final eoncenti tion ot 40 mg/ml
b) Immunization
Ihe fust two immunizations were performed using the toxoid A and toxoid B immunogens described above A total of 3 different immunization combinations were employed I or the first immunization group.02 ml ot toxoid Λ was emulsified in an equal volume ol Tiler Max adjuvant (CvtRx) liter Max was used in oidei to conserve the amount o ifl immunogen used, and to simplify the immunization proceduie This immunization gioup was designated "CTA." For the second immunization group. 0. 1 ml of toxoid B was emulsified in an equal volume of Titer Max adjuvant. This group was designated "CTB." for the third immunization group. 0.2 mi of toxoid A was first mixed with 0.2 ml of toxoid B. and the resulting mixture was emulsified in 0.4 ml of Tiler Max adjuvant. This group was designated "CTAB." In this way . three separate immunogen emulsions were prepared, with each emulsion containing a final concentration of 2.0 mg/ml of toxoid A (CTA) or toxoid B (CTB) or a mixture of 2.0 mg/ml toxoid A and 2.0 mg/ml toxoid B (CTAB).
On day 0. White Leghorn hens, obtained from a local breeder, were immunized as follow s: Group CTA. four hens were immunized, with each hen receiving 200μg of toxoid A. v ia two intramuscular (l .M.) injections of 50μl of CTA emulsion in the breast area.
Group CTB. One hen was immunized with 200μg of toxoid B. via two l.M. injections of 50μl of CTB emulsion in the breast area. Croup CTAB. four hens were immunized, with each hen receiving a mixture containing 200μg of toxoid A and 200μg of toxoid B. via two l . M . injections of l OOμl of CTAB emulsion in the breast area. The second immunization was |ierformed 5 w eeks later, on day 35. exactly as described for the first immunization above.
I n order to determine w hether hens previously immunized with ( '. difficile toxoids could tolerate subsequent booster immunizations using native toxins, a single hen from group CTAB was immunized for a third time, this time using a mixture of the native toxin A and nativ e loxin B described in section (a) above (these toxins were not formaldehyde-treated, and were used in their active form ). T his was done in order lo increase the amount ( liter) and affinity oϊ specific antitoxin antibody produced by the hen over that achieved by immunizing w ith loxoi s only. On day 62. 0. 1 ml of a toxin mixture was prepared which contained 2()0μg of native toxin A and 200μg of native toxin B. T his toxin mixture was then emulsified in 0. 1 ml of filer Max adjuvant. A single CT AB hen was then immunized with the resulting immunogen emulsion, v ia two I.M. injections of l OOul each, into the breast area.
T his hen w as marked with a wing band, and observed for adverse effects for a period of approximately I week, after w hich time the hen appeared to be in good health.
Because the CTAB hen described above tolerated the booster immunization with native toxins A and B with no adverse effects, it was decided to boost the remaining hens with native toxin as well. On day 70. booster immunizations were performed as follow s: Group
CTA. A 0.2 ml volume of the 4 mg/ml native toxin A solution was emulsified in an equal volume of Titer Max adjuvant. Each of the 4 hens was then immunized with 200μg of native toxin A. as described for the toxoid A immunizations above. Group CTB. A 50μl v olume of the 4 mg/ml native toxin B solution was emulsified in an equal volume of Titer Max adjuvant he hen was then immunized with 200μg of native toxin B. as described for the toxoid B immunizations above Group CTAB A 0 15 nil volume of the 4 mg/ml native loxin A solution was first mixed with a 0.1 5 ml volume the 4 mg/ml native toxin B solution The resulting toxm mixture w as then emulsified in 0 3 ml of T iler Max adjuvant T he 3 remaining hens ( the hen with the wing band was not immunized this time ) were then immunized w ith 200μg of native toxin A and 200μg of native toxin B as described for the toxoid A - toxoid B immunizations (CTAB) above On dav 85. all hens received a second booster immunization using native toxins, done exactly as described for the first boost with native toxins above.
All hens tolerated both booster immunizations w ith native toxins wilh no adverse effects Λs previous literature references describe the use ot formaldeh} de-treated toxoids. this is apparently the first time that anv immunizations hav e been performed using nativ e ( diffic ile toxins
e) Purification Of Antitoxins
I ggs were collected from the hen in group CTB 10- 1 2 days follow mg the second immunization w ith toxoid ( day 35 immunization described in section ( b) abov e ), and Irom the hens in groups CT A and CTAB 20-2 1 days following the second immunization w ith toxoid l o be used as a pre-immune ( negative) control, eggs were also collected from unimmunized hens l i om the same flock. Egg yolk immunoglobulin ( IgY7) w as extracted Irom the 4 gioups ol eggs as described in Example 1 (c ). and the final IgY' pellets were solubihzed m the onginal v olk v olume of PBS w ithout thimerosal Importantly , thimerosal was excluded because it would have been toxic to the CHO cells u.sed in the toxin neutralization assav s described m section (d) below
d) Assay Of Toxin Neutralization Activity
T he toxin neutralization activity of the IgY solutions prepared in section (c ) above was determined using an assav sy stem that was modified from published methods | Ehπch et al . Infect. Immun. 28: 1041 - 1043 ( 1992): and McGee et al. Microb Path 12 333-341 ( 1 992). j
Λs additional controls, affinity-purified goat anti-C difficile toxm A ( l ech I ab) and affinity - purified goat anti-C difficile toxin B ( ech Lab) were also assayed for toxin neutralization activity . The IgY solutions and goat antibodies were serially diluted using F 12 medium (GIBCO) which was supplemented with 2% FCS (GIBCO)(this solution will be referred to as "medium" for the remainder of this E.xample). The resulting antibody solutions were then mixed with a standardized concentration of either native ( '. difficile toxin A (Tech Lab), or native ( ' difficile toxin B (Tech Lab), at the concentrations indicated below . Following incubation at 37°C for 60 min.. l OOμl volumes of the toxin ÷- antibody mixtures were added lo the wells of 96-well microtiter plates (Falcon Microtest I II ) which contained 2.5 x 10J Chinese Hamster Ovary (CHO) cells per well (the CT IO cells w ere plated on the previous day to allo them lo adhere to the plate wells). The final concentration of toxin, or dilution of antibodv indicated below refers to the final test concentration of each reagent present in the respectiv e microtiter plate wells. Toxin reference wells were prepared which contained CHO cells and toxin A or toxin B at the same concentration used for the toxin plus antibody mixtures ( these wells contained no antibody). Separate control wells were also prepared w hich contained CHO cells and medium only. The assav plates were then incubated for 1 - 24 hi s. in a 37°C. humidified. 5% CO, incubator. On the following dav. the remaining adherent ( v iable ) cells in the plate w ells were stained using 0.2% crystal violet ( Mallinckrodt) dissolv ed in 2% ethanol. f r 1 0 min. Excess slain w as then removed by rinsing with water, and the stained cells were solubihzed by adding l OOμl of 1 % SDS (dissolved in water) to each w ell. T he absorbance of each well was then measured at 570 nm. and the percent cWotoxicity of each test sample or mixture was calculated using the following formula:
% CHO Cell Cvtotoxicirv = [ 1 - i— S' Sa pl ^ χ lQQ
Abs. Control
Unlike previous reports w hich quantitale results visually by counting cel l rounding by microscopy , this Example utilized spectrophotometric methods to quantitate the ( '. difficile toxin bioassav . In order to determine the toxin A neutralizing activity of the CTA. CTAB. and pre-immune IgY preparations, as well as the affinity-purified goat antitoxin A control, dilutions of these antibodies were reacted against a 0. 1 μg/ml concentration of native loxin A (this is the approx. 50% cytotoxic dose of toxin A in this assay sy stem). The results are show n in Figure 3. Complete neutralization of toxin A occurred with the CTA IgY (antitoxin A. above ) at dilutions of 1 :80 and lower, w hile significant neutralization occurred out to the 1 :320 dilution. The CTAB IgY (antitoxin A + toxin B. above) demonstrated complete neutralization at the 1320-1 160 and lower dilutions, and significant neutralization occurred out to the 1 1280 dilution The commercially available afflnitv-puπfled goat antitoxin A did not completely neutralize toxm A at anv of the dilutions tested, but demonstiated significant neutralization out to a dilution of 1 1.280 Ihe preimmune IgY did not show any toxm A neutralizing activity al anv ot the concentrations tested fhese results demonstiute that IgY purified from eggs laid bv hens immunized with toxin A alone or simultaneously with toxin A and toxin B is an effective toxm A antitoxin
Ihe toxin B neutralizing activity of the CTAB and pie-immune IgY preparations and also the affinity -purified goat antitoxin B control was determined bv reacting dilutions ot these antibodies against a concentration ot native toxin B ol 01 ng/ml (approximatelv the 50% cvtotoxic dose ol toxin B in the assay system) Ihe tesults aie shown in 1 iguie 4
C omplete neutralization of tox B occurred with the C IΛB IgY (antitoxin \ - toxin B above) at the I 40 and lower dilutions and significant neutiahzation occuried out to the 1 320 dilution Ihe ulflnitv-purified goat antitoxin B demonstrated complete neutralization at dilutions ol 1640 and lower, and significant neutralization occurred out to a dilution ot I 2560 Ihe pieimmunc IgY did not show anv toxin B neutralizing activity at anv of the concentrations tested These results demonstrate that IgY purified from eggs laid bv hens immunized simultaneously with toxin A and toxin B is an ettective toxin B antitoxin In a separate study the toxin B neutralizing activity ol C I B C I \B and pre-immune
IgY prepaiations was determined bv leading dilutions ot these antibodies against a native loxin B concenlration ot 01 μg/ml (approximatelv 100% cvtotoxic dose ol toxm B in this assav system) Ihe results aie shown in I igure s
Significant neutralization ot toxm B occurred with the C TB IgY (antitoxin B above) at dilutions ot 180 and lower while the CTAB IgY (antitoxin A » toxin B above) was found to have significant neutralizing activity al dilutions of 140 and lower Ihe pieimmunc IgY did not show toxin B neutiahzing activity at anv ot the eoneentiations tested I hese icsults demonstiate that IgY purified from eggs laid bv hens immunized with toxin B alone oi simultaneously with toxm A and toxin B is an effective toxin B antitoxin EXAMPLE 9
In vivo Protection Of Golden Syrian Hamsters From C difficile Disease Bv Avian Antitoxins Against C difficile Toxins A And B
fhe most extensively used animal model to study C difficile disease is the hamster.
ILverlv et al . Infect. Immun.47:349-352 (1992).) Several other animal models for antibiotic-induced diarrhea exist, but none mimic the human form of the disease as closely as the hamster model. [R. feketv. " Immal Models of Antibioiie-Induced Co iis." in O. Zak and M Sande (eds.). Experimental Models in Antimicrobial Chemotherapy. Vol 2. pp.61-72. ( 1986) I In this model, the animals are first predisposed to the disease by the oral administration of an antibiotic, such as clindamycin. which alters the population of normally- occurring gastrointestinal flora (Feketv. at 61-72). Following the oral challenge of these animals with viable (' difficile organisms, the hamsters develop cecitis. and hemorrhage, uleeration. and inflammation are evident in the intestinal mucosa [Eyerlv et al.. Infect Immun 47349-352 (I985).| The animals become lethargic, develop severe diarrhea, and a high percentage ol them die from the disease. [Lyerly el al Infect. Immun 47349-352 (1985) I This model is therefore ideally suited for the evaluation of therapeutic agents designed for the treatment or prophylaxis of C difficile disease fhe ability oϊ the avian (' difficile antitoxins, described in Example I above, to protect hamsters Irom (' difficile disease was evaluated using the Golden Syrian hamster model of ι difficile infection Ihe E.xample involved (a) preparation ot the avian (' difficile antitoxins, (b) //; vivo protection of hamsters from (' difficile disease by treatment with avian antitoxins, and (e) long-term survival of treated hamsters
a) Preparation Of The Avian C. difficile Antitoxins
Eggs were collected from hens in groups CTA and CTAB described in Example 1 (b) above, fo be used as a pre-immune (negative) control, eggs were also purchased from a iocal supermarket Egg yolk immunoglobulin (IgY) was extracted from the 3 groups of eggs as described in Example I (c). and the final IgY pellets were solubihzed in one fourth the original yolk volume of Ensure R nutritional formula. b) In vivo Protection Of Hamsters Against C. difficile Disease By
Treatment With Avian Antitoxins fhe avian C difficile antitoxins prepared in section (a) above were evaluated toi their ability to protect hamsteis from ( difficile disease using an animal model system which was modified liom published piocedurcs [1 eketv. at 61-72 Boiπelio el al J Med Microbiol
2453-64 (1987) kirn et al Infect Immun .552984-2992 (1987) Borπello a al I Med Microbiol 25191-196 (1988) Delmee and Avesani I Med Microbiol 3385-90(1990) and 1 verlv et al Infect Immun 592215-2218 ( 1991 ) j 1 or the studv three separate experimental groups were used with each group consisting ol 7 female Golden Svπan hamsters (C buries River), approximatelv 10 weeks old and weighing approximately 100 gms each I he three gioups were designated 'CTA " "CI AB" and Pie-immune These designations coiiesponded to the antitoxin preparations with which the animals m each group were tieated I ach animal was housed in an individual cage and was olteied food and vvatet ad libitum through the entire length ol the studv On dav 1 each animal was orallv administered 10 ml ot one ol the three antitoxin preparations (prepared section (a) above) at the following timcpoints 0 hrs 4 hrs and 8 hrs On dav 2 the dav 1 treatment was icpeated On dav 3 at the 0 hr timepoint each animal was again administered antitoxin as desciibed above \t 1 hi each animal was orallv admmisteied 30 mg ol chndamvcin-f 1C1 (Sigma) in 1 ml ol water I his lieatment predisposed the animals to infection with C difficile \s a eontiol lor possible endogenous ( difficile colonization an additional animal liom the same shipment (untreated) was also administered 30 mg of clmdamvcin-HC 1 in the same manner I his clindamycin control animal was left untieated (and unmtccted) toi the lemamdu ol the studv \t the 4 hi and 8 hr timcpoints the animals weie administered antitoxin as desciibed above On dav 4 at the 0 hi timepoint each animal was again administered antitoxin as described above λt 1 hr each animal was oiallv challenged with 1 ml ot C difficile inoculum which contained approx 100 ( difficile strain 43596 organisms in ste le saline ( difficile stiain 43596 which is a serogroup ( sliain was chosen because it is icpiesentative ot one ol the most tiequentlv-occuiπng serogioups isolated Irom patients with antibiotic-associated pseudomembrςinous colitis [Delmee cl al I Cl Microbiol 282210-2214 (1985) | In addition this strain has been pieviouslv demonstrated lo be vuulent in the hamslei model ol infection | Delmee and Avesani I Med Mieiobiol 3385-90 (1990) I \t the 4 hi and 8 hi timcpoints the animals were administered antitoxin as desciibed above On davs 5 through 13 the animals were administered antitoxin 3x per dav as described for day 1 above, and observed for the onset of diarrhea and death. On the morning of day 14. the final results of the study were tabulated. These results are shown in fable 13.
Representative animals trom those that died in the Pre-immune and CTA groups were necropsied Viable ( diffic ile organisms were cultured from the ceca of" these animals, and the gross pathology of the gastrointestinal tracts of these animals was consistent w ith that expected tor ( difficile di.sease ( inflamed, distended, hemorrhagic cecum. filled with watery diarrhea-hke material ) I n addition, the clindamycin control animal remained healthy throughout the entire study period, thereloie indicating that the hamsters used in the study had not prev iously been colonized with endogenous C difficile organisms prior to the start ot the study I ollow g the final antitoxin treatment on day 13. a single surviving animal from the (T \ gi oup. and also irom the C l AB group, w as sacrificed and necropsied No pathology was noted in either animal
TABLE 13
I reatmenl Results
Figure imgf000071_0001
h eatment of hamsters with orally -administered toxin A and toxin B antitoxin ( group C' f \B ) successfully protected 7 out of 7 ( 100%) ol the animals from ( ' diffic ile disease h eatment ol hamsters w ith orally -administered toxin A antitoxin ( group CTA ) protected 5 out of 7 ( 71 %) ol these animals from C difficile disease Treatment using pre-immune IgY was not protectiv e against ( ' difficile disease, as only 1 out of 7 ( 14%) ol these animals surv iv ed These results demonstrate that the avian toxin A antitoxin and the av ian tox A -* toxin B antitoxin effectively protected the hamsters trom ( ' diffic ile disease These results also suggest that although the neutralization of toxin A alone confers some degree of protection against C difficile disease, order to achieve maximal protection, simultaneous antitoxin and antitoxin B activity is necessary
c) Long-Term Survival Of Treated Hamsters
It has been previously reported in the literature that hamsteis treated with oiallv - administered bov ine antitoxin IgG concentrate are protected fro ( ' difficile disease as long as the treatment is continued, but when the treatment is stopped, the animals develop diarrhea and subsequently die within 72 hrs. [Lyeriy et al . Infect. Immun.. 59(6):2215-2218 ( 1991 ).]
In order to determine whether treatment of ( '. difficile disease using avian antitoxins promotes long-term survival following the discontinuation of treatment, the 4 surviving animals in group CTA. and the 6 surviving animals in group CT AB were observed for a period of 1 1 days (264 hrs.) following the discontinuation of antitoxin treatment described in section ( b) above. All hamsters remained healthy through the entire post-treatment period. This result demonstrates that not only does treatment with avian antitoxin protect against the onset of C difficile disease (i. e.. it is effective as a prophylactic ), it also promotes long-term surviv al beyond the treatment period, and thus provides a lasting cure.
EXAMPLE 10
In vivo Treatment Of Established C. difficile I nfection In Golden Syrian Hamsters With Avian Antitoxins Against C difficile T oxins A And B
fhe ability of the avian ( '. difficile antitoxins, described in Example 8 above, to treat an established ( ' difficile infection w as evaluated using the Golden Syrian hamster model. The Example involved (a) preparation of the avian C. difficile antitoxins, (b) in vivo treatment of" hamsters with established ( ' difficile infection, and (c ) histologic evaluation of cecal tissue.
a) Preparation Of The Avian C. difficile Antitoxins
Eggs were collected from hens in group CTAB described in Example 8 ( b) above. which were immunized with ( '. difficile toxoids and native toxins A and B. Eggs purchased from a local supermarket w ere used as a pre-immune ( negative) control. Egg y olk immunoglobulin ( IgY ) was extracted from the 2 groups of eggs as described in Example 1 (e ). and the final IgY pellets were solubihzed in one-fourth the original yolk volume of Ensure1.'* nutritional formula.
b) In vivo Treatment Of Hamsters With Established C. difficile Infection
The avian ( '. difficile antitoxins prepared in section ( a ) above were evaluated for the ability to treat established ( '. difficile infection in hamsters using an animal model system which was modified from the procedure which was described for the hamster protection study in Example 8(b) above
Tor the study, four separate experimental groups were used, with each group consisting ol 7 female Golden Syrian hamstei s (Charles River), approx 10 weeks old. weighing approximatelv 100 gms. each Each animal was housed separately , and was offered food and w ater ad libitum through the entire length of the study
On day I ot the study, the animals in all four groups were each predisposed to C diffic ile infection bv the oral administration of 3 0 mg of clmdamy cin-HCl (Sigma) in 1 ml ol water On day 2. each animal in all four gioups was orally challenged with 1 ml ol
( diffic ile inoculum, w hich contained approximately 1 0 C diffic ile strain 43596 organisms in ster ile saline ( difficile strain 43596 was chosen because it is representative ol one of the most ti equentlv -occurnng serogroups isolated from patients with antibiotic-associated pseudomembranous colitis [Delmee et al . .1 Clin. Microbiol . 28.2210-2214 ( 1990) ] In addition, as this w as the same ( diffic ile strain used in all of the previous [ xamples abov e, it w as again used in ol der to prov ide experimental continuity
On 3 of the study (24 hi s post-infection), treatment w as started f oi tw o of the tour groups ol animals Each animal of one group was orally administered I 0 ml of the CTAB IgY' preparation (prepaicd in section (a) above) at the following timepoints 0 hrs . 4 hrs . and 8 hi s I he animals in this group were designated "CT AB-24 " The animals in the second group were each orally administered 1 0 ml of the pre-immune IgY preparation (also piepai ed in section ( a ) above ) at the same timepoints as f oi the C I ΛB gr oup 1 hese animals wer e designated "Pιe-24 " Nothing was done to the remaining two groups ol animals on day
On day 4. 48 hi s post-infection, the treatment described tor day 3 above w as l epeated for the CTΛB-24 and Pre-24 groups, and was initiated for the remaining two gioups at the same timepoints I he final two groups of animals were designated "CTΛB-48" and "Pιe-48" respectively
On dav s 5 through 9. the animals in all four gioups were administered antitoxin oi pre-immune IgY'. 3\ per day . as described foi day 4 above T he four expenmental gioups are summarized in fable 14 TABLE 14
Experimental I reatment Groups
Figure imgf000074_0001
All animals were observed for the onset ot diarrhea and death through the conclusion ol the study on the morning of day 10 fhe results ol this study aie displayed in Table 15
TABLE 15
F xpeπmental Outcome— av 10
Figure imgf000074_0002
Eighty-six percent ot the animals which began leceiving tieatment with antitoxin IgY' at 24 his post-infection (CTAB-24 above) survived, while 57% ot the animals treated with antitoxin IgY starting 48 hrs post-infection (CTAB-48 above) suivived In contrast, none ol the animals leceiving pre-immune IgY starting 24 hrs post-intection (Pre-24 above) survived, and onlv 29% ol the animals which began receiving tieatment with pre-immune IgY at 48 hrs post-infection (Pre-48 above) survived through the conclusion of the studv I hese results demonsti te that avian antitoxins laised against C difficile toxins A and B aie capable ol successfully tieatinsi established C difficile infections in vivo
c) Histologic Evaluation Of Cecal Tissue
In older to further evaluate the ability ot the IgY preparations tested in this study to neat established C difficile infection, histologic evaluations we e pertormed on cecal tissue specimens obtained liom representative animals fiom the study described in section (b) above
Immediately following death, cecal tissue specimens were removed liom animals which died in the Pιe-24 and Pιe-48 groups I ollowmg the completion ol the study, a lepiesentative suivivmg animal was sacrificed and cecal tissue specimens were removed from the CTAB-24 and CTAB-48 groups. A single untreated animal from the same shipment as those used in the study was also sacrificed and a cecal tissue specimen was removed as a normal control. All tissue specimens were fixed overnight at 4°C in 10% buffered formalin. The fixed tissues were paraffin-embedded, sectioned, and mounted on glass microscope slides. T he tissue sections were then stained using hematoxylin and eosin (H and E stain), and were examined by light microscopy.
Upon examination, the tissues obtained from the CTAB-24 and CTAB-48 animals show ed no pathology, and were indistinguishable from the normal control. This observation provides further evidence for the ability of avian antitoxins raised against ( '. difficile toxins A and B to effectively treat established C. difficile infection, and to prevent the pathologic consequences which normally occur as a result of C. difficile disease.
In contrast, characteristic substantial mucosal damage and destruction w as observed in the tissues of the animals from the Prc-24 and Pre-48 groups which died from ( '. difficile disease. Normal tissue architecture was obliterated in these two preparations, as most of the mucosal layer w as observed to have sloughed away, and there were numerous large hemorrhagic areas containing massive numbers of erythrocytes.
EXAMPLE 1 1
Cloning And Expression Of C. difficile Toxin A fragments
fhe toxin A gene has been cloned and sequenced. and shown to encode a protein of predicted MW of .308 kd. [Dove et al . Infect. Immun.. 58:480-488 ( 1990). | Given the expense and difficulty of isolating native toxin A protein, it would be advantageous to use simple and inexpensive procaryotic expression systems to produce and purify high levels of recombinant toxin A protein for immunization purposes. Ideally, the isolated recombinant protein would be soluble in order to preserve native antigenicity. since solubihzed inclusion body proteins often do not fold into native conformations. To allow ease of purification, the recombinant protein should be expressed to levels greater than 1 mg/liter of E. coli culture.
To determine whether high levels of recombinant toxin A protein can be produced in E. coli. fragments of the toxin A gene were cloned into various prokaryotic expression v ectors, and assessed for the ability to express recombinant toxin A protein in E. coli. Three prokaryotic expression systems were utilized. These systems were chosen because they drive expression of either fusion (pMALc and pGEX2T) or native ( pET23a-c ) protein to high levels in E. coli. and allow affinity purification of the expressed protein on a ligand containing column. Fusion proteins expressed from pGEX vectors bind glutathione agarose beads, and are eluted with reduced glutathione. pMAL fusion proteins bind amylose resin, and are eluted with maltose. A poly-histidine tag is present at either the N-terminal (pETT 6b) or C-terminal (pET23a-c ) end of pET f usion proteins. This sequence specifically binds NL chelate columns, and is eluted w ith imidazoie salts. Extensive descriptions of these vectors are available [ Williams ei al. ( 1995) DNA Cloning 2: Expression Systems. Glover and Hames. eds. I L Press. Oxford, pp. 1 5-58], and will not be discussed in detail here. The Example involved (a) cloning of the toxin A gene, ( b) expression of large fragments of toxin A in various prokaryotic expression systems, (c) identification of smaller toxin A gene fragments that express efficiently in /;. coli. (d) purification of recombinant toxin A protein by affinity chromatography. and (e) demonstration of functional activity of a recombinant fragment of the toxin A gene.
a) Cloning Of The Toxin A Gene
A restriction map of the toxin A gene is shown in Figure 6. fhe encoded protein contains a carboxy terminal ligand binding region, containing multiple repeats of a carbohydrate binding domain, ( von Eichel-Streiber and Sauerborn. Gene 96: 107- 1 1 3 ( 1990).] The toxin A gene was cloned in three pieces, by using either the polymerase chain reaction ( PCR) to amplify specific regions, ( regions 1 and 2. f igure 6) or by screening a constructed genomic library for a specific toxin A gene fragment ( region 3. f igure 6). T he sequences of the utilized PCR primers arc P I : 5 " GGΛAATT TAGCTGCΛGCΛTCT GΛC 3 ' ( SEQ I D NO. : l ): P2: 5' TCTAGCAAATTCGCTTGT GTTGAA 3' (SEQ ID NO. :2 ): P3 : 5' CTCGC A TATΛGCATTAGACC 3 " (SEQ ID NO.:3 ): and P4: 5' C'fAT CTΛGGCCTAAAGTAT 3' ( SEQ IE) NO.:4). These regions were cloned into prokaryotic expression vectors that express either fusion (pMALc and pGEX2T) or native (pET23a-c) protein to high levels in E. coli. and allow affinity purification of the expressed protein on a ligand containing column.
Clostridium difficile VPI strain 10463 was obtained from the AT CC ( ATCC #43255 ) and grown under anaerobic conditions in brain-heart infusion medium ( BBL). High molecular-weight C difficile DNA was isolated essentially as described by Wren and Tabaqchali ( 1987) .1. Clin. Microbiol.. 25:2402. except proteinase K and sodium dodecyl sulfate (SDS) was used to disrupt the bacteria, and cetyltrimethylammonium bromide precipitation [as described in Ausubel et al.. Current Protocols in Molecular Biology ( 1989)] was used to remove carbohydrates from the cleared lysate. The integrity and yield of genomic DNA was assessed by comparison with a serial dilution of uncut lambda DNA after electrophoresis on an agarose gel. fragments 1 and 2 were cloned by PCR. utilizing a proofreading thermostable DNA polymerase ( native /?/// polymerase; Stratagene). The high fidelity of this polymerase reduces the mutation problems associated with amplification by error prone polymerases (e.g. , Tacj polymerase). PCR amplification was performed using the indicated PCR primers (Figure 6) in 50 μl reactions containing 10 mM Tris-HCI(8.3), 50 mM KC1. 1 .5 mM MgCE. 200 μM each dNTP. 0.2 μM each primer, and 50 ng C difficile genomic DNA. Reactions were overlaid w ith 1 00 μl mineral oil. heated to 94°C for 4 min. 0.5 μl native /;/// polymerase ( Stratagene) added, and the reaction cycled 30x at 94°C for I min. 50°C for 1 min. 72°C for 4 min. followed by 10 min at 72°C. Duplicate reactions were pooled, chloroform extracted, and ethanol precipitated. After washing in 70% ethanol. the pellets were resuspended in 50 μl TE buffer [ 10 mM Tris-HCL. 1 mM EDTA pH 8.0]. Aliquots of 10μl each were restriction digested w ith either Eco MHi cW ( fragment 1 ) or EcoRllPstl ( fragment 2). and the appropriate restriction fragments were gel purified using the Prep-A-Gene kit ( BioRad). and ligated to either Λ'cuRI/.SΗωl-restricted pGEX2T (Pharmacia) v ector ( fragment I ), or the EcoRl/Psll pMAlc (New England Biolabs) vector ( fragment 2). Both clones are predicted to produce in-frame fusions with either the glutathione-S-transfcrase protein (pGFX v ector) or the maltose binding protein ( pMAL vector). Recombinant clones were isolated, and confirmed by restriction digestion, using standard recombinant molecular biology techniques. I Sambrook et al.. Molecular Cloning. A Laboratory Manual ( 1989). and designated pGA30- 660 and pMΛ660- l 100. respectiv ely (see figure 6 for description of the clone designations). ] f ragment 3 was cloned from a genomic library of size selected PstI digested
( '. difficile genomic DNA. using standard molecular biology techniques (Sambrook et al. ). Given that the fragment 3 internal Psl\ site is protected from cleavage in C. difficile genomic DNA [ Price et al.. Curr. Microbiol.. 16:55-60 ( 1987)]. a 4.7 kb fragment from PstI restricted ( '. difficile genomic DNA was gel purified, and ligated to Psi] restricted, phosphatase treated pUC9 DNA. The resulting genomic library was screened with a oligonucleotide primer- specific to fragment 3. and multiple independent clones were isolated. The presence of fragment 3 in several of these clones was confirmed by restriction digestion, and a clone of the indicated orientation ( figure 6) was restricted with Bam\\\IHind\ . the released fragment purified by gel electrophoresis. and ligated into similarly restricted pET23c expression vector DNA (Novagen). Recombinant clones were isolated, and confirmed by restriction digestion. This construct is predicted to create both a predicted in frame fusion with the pE T protein leadei sequence, as well as a predicted C-terminal pol -histidine alfinity tag. and is designated pPAl 100-2680 (see figure 6 for the clone designation)
b) Expression Of Large Fragments Of Toxin A In E. coli
Piotein expression from the three expression constructs made in (a) was induced, and analyzed by Western blot analysis with an affinity purified, goat polycional antiserum directed against the toxm A toxoid ( lech Lab). The procedures utilized for protein induction. SDS-
PAGE. and Western blot analysis are described in detail in Williams el al (1995). supra In brief.5 ml 2X Y1 (16 g tryptone. 10 g yeast extract.5 g NaC per liter, pll 75 +- 100 μg/ml ampiciilin were added to cultures of bacteria (BL21 lor pMAl and pGI X plasmids. and BL2KD13)[ ysS for pL I plasmids) containing the appropriate recombinant clone which were induced to express recombinant protein by addition of IPTG to 1 mM Cultuies were grown al 37°C. and induced when the cell density reached 05 ODMll) Induced protein was allowed to accumulate tor two his alter induction Protein samples were prepared bv pelleting 1 ml ahquots ol bacteria by centrifugation (I mm in a microfuge). and resuspension ot the pelleted bacteria in 150 μl ol 2x SDS-PAGE sample buffer [Williams el al (1995). supia Ihe samples weie heated to 95°C for 5 m . the cooled and 5 or 10 μl ahquots loaded on 75%
SDS-PAGE gels BioRad high molecular weight protein markers were also loaded, to allow estimation of the MW of identified lusion proteins After cTecliophoiesis. protein was delected either generally by staining gels with Coomassie blue, oi specifically, by blotting to nitrocellulose lor Western blot detection ol specific immunoieactive protein Western blots. (performed as described in I xample 3) which detect toxin A reactive piotein in cell lysates ot induced protein from the three expression constructs are shown in 1 igure 7 In this figure, lanes 1-3 contain cell lysates piepared from / coli strains containing pPAl 100-2860 in B!21(DL3)lysE cells: lanes 4-6 contain cell lysates piepared from L coli stiains containing pPAl 100-2860 in B12l(DL3)ly sS cells, lanes 7-9 contain cell h sales prepared from h eo strains containing pMA30-660. lanes 10-12 contain cell lysates prepared liom L coli strains containing pMA660-l 100 fhe lanes were probed with an affinity purified goat antitoxin A polycional antibody (lech Lab). Control lysates from uninduccd cells (lanes 1.7. and 10) contain very little immunoreactive material compared to the induced samples in the remaining lanes. The highest molecular weight band observed for each clone is consistent with the predicted size of the full length fusion protein.
Each construct directs expression of high molecular weight HMW) protein that is reactive with the toxin A antibody. The size of the largest immunoreactive bands from each sample is consistent with predictions of the estimated MW of the intact fusion proteins. This demonstrates that the three fusions are in-frame, and that none of the clones contain cloning artifacts that disrupt the integrity of the encoded fusion protein. However, the Western blot demonstrates that fusion protein from the two larger constructs ( pGA30-660 and pPA l 1 00- 2680) are highly degraded. Also, expression levels of toxin A proteins from these two constructs are low. since induced protein bands are not visible by Coomassie staining (not shown ). Several other expression constructs that fuse large sub-regions of the toxin A gene to either pMALc or pET23a-c expression vectors, were constructed and tested for protein induction. These constructs were made by mixing gel purified restriction fragments, derived I rom the expression constructs shown in figure 6. with appropriately cleaved expression v ectors, ligating. and selecting recombinant clones in which the toxin A restriction fragments had ligated together and into the expression vector as predicted for in-frame fusions. The expressed toxin A interval within these constructs are shown in Figure 8. as well as the internal restriction sites utilized to make these constructs.
As used herein, the term "interval" refers to any portion (i. e.. any segment of the toxin which is less than the whole loxin molecule) of a clostridial toxin. In a preferred embodiment, "interval" refers to portions of C difficile toxins such as toxin A or toxin B. It is also contemplated that these intervals will correspond to epitopes of immunologic importance, uch as antigens or immunogens against which a neutralizing antibody response is effected. It is not intended that the present invention be limited to the particular intervals or sequences described in these Examples. It is also contemplated that sub-portions of intervals
(e.g.. an epitope contained within one interval or which bridges multiple intervals) be used as compositions and in the methods of the present invention.
In all cases. Western blot analysis of each of these constructs with goat antitoxin A antibody ( Tech Lab) detected HMW fusion protein of the predicted size ( not shown ). This confirms that the reading frame of each of these clones is not prematurely terminated, and is fused in the correct frame with the fusion partner. However, the Western blot analysis revealed that in ail cases, the induced protein is highly degraded, and. as assessed by the absence oϊ identifiable induced protein bands by Coomassie Blue staining, are expressed only at low levels. These results suggest that expression of high levels of intact toxin A recombinant protein is not possible when large regions of the toxin A gene are expressed in E. coli using these expression vectors.
e) High Level Expression Of Small Toxin A Protein Fusions In
E. coli
Experience indicates that expression difficulties are often encountered when large (greater than 100 kd) fragments are expressed in /". coli. A number of expression constructs containing smaller fragments of the toxin A gene were constructed, to determine if small regions of the gene can be expressed to high levels without extensive protein degradation. A summary of these expression constructs are shown in figure 9. All were constructed by in- frame fusions of convenient toxin A restriction fragments to either the pMALc or pET23a-c v ectors. Protein preparations from induced cultures of each of these constructs were analyzed by both Coomassie Blue staining and Western analysis as in (b) above. In all cases, higher levels of intact, full length fusion proteins were observed than with the larger reeombinants from section ( b).
(I) Purification Of Recombinant Toxin A Protein
Large scale (500 ml) cultures of each recombinant from (e ) were grown, induced, and soluble and insoluble protein fractions were isolated. The soluble protein extracts were affinity chromatographed to isolate recombinant fusion protein, as described [ Williams el al. ( 1 994 ). supra]. In brief, extracts containing tagged pET fusions were chromatographed on a nickel chelate column, and eluted using imidazoie salts as described by the distributor (Novagen). Extracts containing soluble pMAL fusion protein were prepared and chromatographed in column buffer ( 10 mM NaP0 , 0.5M NaCl. 10 mM -mercaptoethanol. pH 7.2) over an amylose resin column (New England Biolabs). and eluted with column buffer containing 10 mM maltose as described [Williams et al. ( 1995 ). supra]. When the expressed protein was found to be predominantly insoluble, insoluble protein extracts were prepared by the method described in E xample 1 7. infra. T he results are summarized in fable 1 6. f igure 10 shows the sample purifications of recombinant toxin A protein, in this figure, lanes I and 2 contain MBP fusion protein purified by affinity purification of soluble protein. TABLE 16
Purification Of Recombinant Toxin A Protem
Figure imgf000081_0001
pP pCT23 vecloi . pM-pMAL c vector A=toxιn A '" Based on I 5 OD,,,, - I mg/ml (extinction coefficient ot MBP)
' ' I stimated bv Coomassie staining of SDS-PAGE gels
Lanes 3 and 4 contain MBP fusion protein purified by solubihzation of msoluble inclusion bodies The purified fusion protein samples are pMA 1870-2680 ( lane 1 ). pMΛ660- l 100 (lane 2 ). pMA30ϋ-600 ( lane s ) and pMΛ 1450- 1870 (lane 4)
Poor y ields ot af finity purified protein were obtained when poly-histidine tagged pET v ectors were used to drive expression (pPAl 100- 1450. pP l 100- 1870) Howevei . significant piotein yields were obtained from pMAL expression constructs spanning the entire toxin A gene, and yields of full-length soluble lusion protein ranged fiom an estimated 200-400 μg/500 ml culture (pMA30-300) to greater than 20 mg/500 ml culture (pMA 1610- l 870) Only one interval was expressed to high levels as strictly insoluble protein (pMΛ300-660) I hus. although high level expression was not observed when using large expression constructs tr om the toxin A gene, usable lev els of recombinant protein spanning the entire toxm A gene were obtainable by isolating induced protein from a series of smaller pMAI expression constructs that span the entire toxin A gene. This is the first demonstration ol the feasibility ol expressing recombinant toxin A protem to high levels in E c oli c) Hemagglutination Assay Using The Toxin A Recombinant
Proteins
T he carboxy terminal end consisting of the repeating units contains the hemagglutination activity or binding domain of C difficile toxin A. To determine whether the expressed toxin A recombinants retain functional activ ity, hemagglutination assays were performed. Two toxin A recombinant proteins, one containing the binding domain as either soluble affinity purified protein (pMA 1870-2680) or SDS solubihzed inclusion body protein ( pPA l 870-2680) and soluble protein from one region outside that domain (pMA I 100- 1610) were tested using a described procedure. [H.C. rivan et. al . Infect. Immun.. 53:573 ( 1986). I t itrated rabbit red blood cells ( RRBO(Cocalico) were washed several times with
Tris-bu fer ( 0. 1 M Tris and 50 mM NaCl ) by centrifugation at 450 x g for 10 minutes at 4° C. A 1 % RRBC suspension was made from the packed cells and resuspended in T ris-buffer. Dilutions of the recombinant proteins and native toxin A ( T ech Labs) were made in the T ris- buffer and added in duplicate to a round-bottomed 96-well microtiter plate in a final volume of 1 00 μl. To each w ell. 50 μl of the 1 % RRBC suspension was added, mixed by gentle tapping, and incubated at 4°C for 3-4 hours. Significant hemagglutination occurred only in the recombinant proteins containing the binding domain ( pMA 1 70-2680) and native toxin A. The recombinant protein outside the binding domain (pMA 1 1 0- 161 ) displayed no hemagglutination activity. Using equivalent protein concentrations, the hemagglutination titer for toxin A w as 1 :256. while tilers for the soluble and insoluble recombinant proteins of the binding domain were 1 :256 and about 1 :5000. Clearly, the recombinant proteins tested retained functional activity and w ere able to bind RRBC's.
EXAMPLE 12 functional Activity Of IgY Reactive Against Toxin A Recombinants
T he expression oϊ recombinant toxin A protein as multiple fragments in E.coli has demonstrated the feasibility of generating toxin A antigen through use of recombinant methodologies ( E.xample 1 1 ). The isolation of these recombinant proteins allows the immunoreactivity of each individual subregion of the toxin A protein to be determined ( i. e.. in a antibody pool directed against the native toxin A protein). T his identifies the regions ( if any) for which little or no antibody response is elicited when the w hole protein is u.sed as a immunogen. Antibodies directed against specific fragments of the toxin A protein can be purified bv affmitv chromatography against recombinant toxin A protein, and tested foi neutralization ability This identifies anv toxm A subregions that are essential foi producing neutralizing antibodies Comparison with the levels of immune response directed against these intervals when native toxin is used as an immunogen predicts whether potentially higher 5 liters ot neutralizing antibodies can be produced by using recombinant protein directed against a individual tegion lather than the entire protein I inallv since it is unknown whether antibodies leuctive to the recombinant toxin A proteins produced in 1 xample 11 neutiahze toxin A as effectively as antibodies laised against native toxm Λ (Examples 9 and 10) the protective ability oi a pool ot antibodies affinity purified against recombinant toxin A
10 tiagments was assessed foi its ability to neutralize toxin A
This Example involved (a) epitope mapping of the toxm A protem lo determine the titie ol specific antibodies directed against individual subregions ot the toxin A protein when native toxm v piotein is used as an immunogen. (b) affinity purification of IgY leactive against iecombinant proteins spanning the toxin A gene (e) loxin A neutralization assays with
I ^ alllnitv puiilled IgY leactive to recombinant toxin A protein to identify subiegions of the toxin A piotein that induce the production ot neutralizing antibodies, and deteimmation ot whether complete neutralization of toxin A can be elicited with a mixtuie ol antibodies icaclive to iecombinant toxin A protein
0 a) Epitope Mapping Of The Toxin A Gene
Ihe alfinity purification ol iecombinant toxin A protein specific to defined intervals ol the loxin \ piotein allows epitope mapping of antibody pools duected against native toxm \ I his has not pieviouslv been possible since previous expression ol toxm A recombinants has been assessed onlv bv Western blot analysis, without knowledge ol the expression levels ot 5 the piotem ft' g von I ichel-Slreiber el al 1 den Microbiol .13555-64 ( 1989)) I hus. high oi low teuctivitv ot iecombinant toxm A protein on Western blots mav reflect protem expression level differences not immunoreactivity differences (men that the purified iecombinant piotein generated in Lxample 1 I have been quuntitated the issue of lelutive immunoieactiv itv ot individual legions of the toxin A piotein was precisely addtesscd
30 I oi the purposes ol this I xample. the toxin A protein was subdivided into 6 inteivals
(1-6). numbered Irom the ammo (interval 1) to the caiboxvl (interval 6) termini
Ihe iecombinant proteins coiresponding to these intervals were liom expiession clones (see 1 xample I 1(d) foi clone designations) pMA30-3()0 (interval 1) pMA300-660 (interval 2). pMA660-l 100 (interval 3). pPAl 100-1450 (interval 4). pMA1450-1870 (interval 5) and pMA 1870-2680 (interval 6) These 6 clones were selected because they span the entire protein trom amino acids numbered 30 through 2680. and subdivide the protein into 6 small intervals Also, the carbohydrate binding repeat interval is contained specifically in one interval (interval 6). allowing evaluation of the immune response specifically directed against this region Western blots of 75% SDS-PAGE gels, loaded and electrophoresed with defined quantities of each iecombinant protein, were probed with either goat antitoxin A polycional antibody ( lech Lab) or chicken antitoxin A polycional antibody [pC'IΛ IgY. I xample 8(c)] The blots were prepared and developed with alkaline phosphatase as previously described I Williams el al (1995). supra] At least 90% of all reactivity, in either goat oi chicken antibody pools, was found to be directed against the ligand binding domain (interval 6) The remaining immunoreactivity was directed against all five remaining intervals, and was similar in both antibody pools, except that the chicken antibody showed a much lower reactivity against interval 2 than the goat antibody Ibis clearly demonstrates that when native toxin A is used as an immunogen goats oi chickens, the bulk ot the immune icsponse is directed against the ligand binding domain of the protein, ith the remaining response distributed throughout the remaining 2'3 ot the protein
b) Vffinitλ Purification Of IgY Reactive Against Recombinant
Toxin A Protein
Alfinity columns, containing recombinant toxm A protein Irom the 6 defined mteivals in (a) above, were made and used to (i) affinity purity antibodies teactive lo each individual interval from the CIA IgY pieparation |I xample 8(c) f. and (π) deplete interval specific antibodies from the CTA IgY preparation Affinity columns were made by coupling 1 ml ot
PBS-washed Λctigel resin (Sterogene) with region specific protein and 1/10 final volume oϊ Aid-coupling solution (1M sodium cy noborohydπde) Ihe total icgion specific protein added to each leaction mixture was 27 mg (interval 1).3 mg (intervals 2 and 3).01 mg (interval 4).02 mg (inteival 5) and 4 mg (interval 6) Protein tor intervals 1.3. and 6 was alfinity purified pMAI lusion protein in column butler (see L.xamplc I I) Interval 4 was affinity purified poly -histidine containing pET fusion in PBS. intervals 2 and 5 were Irom inclusion body preparations ol msoluble pMAI. tusion protein, dialyzed extensively in PBS Ahquots of the supernatants from the coupling reactions, betore and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, in all cases greater than 50% coupling efficiencies were estimated. The resins were poured into 5 ml BioRad columns, washed extensively with PBS. and stored at 4°C.
Aliquots of the CTA IgY polycional antibodv preparation were depleted for each individual region as described below. A 20 ml sample of the CTA IgY preparation ( Example
8( e ) | was dialyzed extensiv ely against 3 changes of PBS ( I liter for each dialvsis). quantitated by absorbance at OD-,s„. and stored at 4°C. Six 1 ml aliquots of the dialyzed IgY preparation w ere removed, and depleted individually for each of the six intervals. Each 1 ml aliquot was passed ov er the appropriate affinity column, and the eluate twice reapplied to the column. 1 he eluate was collected, and pooled with a I ml PBS wash. Bound antibody was eluted from the column by w ashing with 5 column volumes of 4 M Guanidine-HCl (in 10 mM T'ris- I ICI. pl l 8.0). T he column was reequilibrated in PBS. and the depleted antibody stock reapplied as described above, The eluate was collected, pooled w ith a 1 ml PBS wash, quantitated by absorbance at D->S(1. and stored at 4° ('. In this manner. 6 aliquots of the CTA IgY preparation were individually depicted for each of the 6 toxin A intervals, by two rounds of affinity depletion. The specificity of each depleted stock was tested by Western blot analysis. Multiple 7.5% SDS-PAGE gels were loaded with protein samples corresponding to all 6 toxin A subregions. After electrophoresis. the gels were blotted, and protein transfer confirmed by Ponceau S staining [protocols described in Williams el al. ( 1995). supra]. After blocking the blots 1 hr at 20°C in PBS+ 0. 1 % Tween 20 ( PBST) containing 5% milk (as a blocking buffer). 4 mi of either a 1 /500 dilution of the dialyzed CTA IgY preparation in blocking buffer, or an equivalent amount of the six depleted antibodv stocks ( using OD:sπ to standardize antibodv concentration ) were added and the blots incubated a further 1 hr at room temperature. The blots were washed and developed with alkaline phosphatase ( using a rabbit anti-chicken alkaline phosphate conjugate as a secondary antibody) as previously described
[ Williams el al. ( 1995). supra]. In all cases, only the target interval was depleted for antibody reactivity , and at least 90% of the reactivity to the target intervals was specifically depleted. Region specific antibody pools were isolated by affinity chromatography as described below, Pen mis of the dialyzed CTA IgY preparation were applied sequentially to each affinity column, such that a single 10 ml aliquot was used to isolate region specific antibodies specific to each of the six subregions. The columns were sequentially washed with 1 0 volumes of PBS. 6 volumes of BBS-Tween. 10 volumes of TBS. and eluted with 4 l Λctisep elution media (Sterogene). T he eluate was dialyzed extensively against several changes of PBS. and the affinity purified antibody collected and stored at 4°C. The volumes of the eluate increased to greater than 10 mis during dialysis in each case, due to the high viscosity of the Actisep elution media. Aliquots of each sample were 20x concentrated using Centricon 30 microconcentrators (Amicon) and stored at 4°C. The specificity of each region specific antibody pool was tested, relative to the dialyzed CTA IgY preparation, by Western blot analysis, exactly as described above, except that 4 ml samples of blocking buffer containing 100 μl region specific antibody (unconcentrated ) were used instead of the depleted CTA IgY preparations. Each affinity purified antibody preparation was specific to the defined interval, except that samples purified against intervals 1 -5 also reacted with interval 6. This may be due to non-specific binding to the interval 6 protein, since this protein contains the repetitiv e ligand binding domain which has been show n to bind antibodies nonspeciflcallv. I Lyerly et al . Curr. Microbiol.. 19:303-306 ( 1989). ] fhe reactivity of each affinity purified antibody preparation to the corresponding proteins was approximately the same as the reactivity of the 1/500 diluted dialyzed CTA IgY preparation standard. Given that the specific antibody stocks were diluted 1 /40. this would indicate that the unconcentrated affinity purified antibody stocks contain 1 / 1 0- 1 /20 the concentration of specific antibodies relative to the starting CT A IgY preparation.
c) Toxin A Neutralization Assay Using Antibodies Reactive Toward Recombinant Toxin A Protein fhe ( IO toxin neutralization assay [Example 8(d)] was used to assess the ability of the depleted or enriched samples generated in ( b) above to neutralize ihe ey totoxicity of toxin A. The general ability of affinity purified antibodies to neutralize toxin A w as assessed by mixing together aliquots of all 6 concentrated stocks of the 6 affinity purified samples generated in (b) above, and testing the ability of this mixture to neutralize a toxin A concentration of 0. 1 μg/ml. The results, shown in figure 1 I . demonstrate almost complete neutralization of toxin A using the affinity purified ( AP) mix. Some epitopes w ithin the recombinant proteins utilized for affinity purification were probably lost when the proteins were denatured before affinity purification [by Guanidine-HCl treatment in ( b) above]. T hus. the neutralization ability of antibodies directed against recombinant piotein is probably underestimated using these affinity purified antibody pools. T his experiment demonstrates that antibodies reactive to recombinant toxin A can neutralize eytotoxicity, suggesting that neutralizing antibodies may be generated bv using recombinant toxm A protein as immunogen
In view ot the observation that the recombinant expiession clones of the toxin A gene divide the protein into 6 subregions the neutralizing ability ot antibodies duected against each ■v individual region was assessed Ihe neutralizing ability ol antibodies directed against the ligand binding domain of toxin A was determined first
In the toxin neutralization experiment shown in Figure 11 interval 6 specific antibodies (mteival 6 contains the ligand binding domain) were depleted horn the dialvzed PI G piepaiation and the ellect on toxin neutralization assayed Inteivul 6 antibodies weie 0 depleted either bv utilizing the interval 6 depleted CTA. IgY preparation trom (b) above ('-6 all depleted I iguie II) or bv addition ot interval 6 protein to the CTA lg\ preparation (estimated to be a 10 fold molai excess over anti-interval 6 immunoglobulin piesent in this piepaiation) to competitively compete foi interval 6 piotein ( 6 piot depleted in figure I 1) In both instances iemoval ol interval 6 specific antibodies reduces the neutiahzation efficiency lelalive lo the starting CT \ IgY prepaiation I his demonstrates that antibodies duected against interval 6 contribute to toxin neutralization Since interval 6 coiiesponds to the ligand binding domain ot the piotem these lesults demonstrate that antibodies duected against this region in the PI G prepaiation contribute to the neutralization ol toxm A in this assay However it is significant that alter removal ol these antibodies the PI G pieparation 0 tetains significant ability to neutralize toxm A (Figure 1 I) I his neutralization is probablv due to the action of antibodies specific to other legions ot the toxin A protein since at least 90" o ol th Inland b diim icgion reactive antibodies were removed m the depleted sample piepaied in (b) above I his conclusion was supported bv comparison of the toxm neutiahzation ol the affinity pun fled (AP) mix compared to affinity purified interval 6 ^ antibodv alone Although some neutralization ability was observed with AP interval 6 antibodies alone the neutralization was significantly less than that observed with the mixture ol all 6 \P antibodv stocks (not shown)
Given that the mix ol all six affinity purified samples almost completely neutralized the evlotoxicilv ol toxin Λ (I iguie 1 I) the relative impoitance of antibodies duected against 0 toxin A intervals 1-5 within the mixture was determined This was assessed in two wavs
Tirst samples containing afllnitv punfled antibodies representing 5 of the 6 intervals weie piepaied such that each individual region was depleted Irom one sample I igure 12 demonstiules a sample neutiahzation cuive comparing the neutiahzation ability ol affinity purified antibody mixes without inteival 4 (-4) or 5 (-5) specific antibodies ielative to the mix ol all 6 affinity purified antibodv stocks (positive control) While the removal of interval 5 specific antibodies had no effect on toxin neutralization (oi mteivuls 1-3 not shown) the loss ol interval 4 specific antibodies significantly reduced toxin neutiahzation (I igure 12) Similar tesults were seen in a second experiment in which uffimitv purified antibodies directed against a single legion were added to interval 6 specific antibodies and the effects on toxin neutiahzation assessed Only interval 4 specific antibodies significantly enhanced neutiahzation when added to inteival 6 specific antibodies (1 iguie 13) fhese results demonstiute that antibodies directed against interval 4 (coi responding to clone pPAl 100-1450 in I iguie 9) aie important foi neutralization ol cvtotoxicitv in this assav I pitope mapping has shown that only low levels ot antibodies reactive to this region aie generated when native toxin A is used as an immunogen [f xample 12(a) | It is hypothesized that immunization with iecombinant piotein specific to this interval will elicit higher tilers oi neutiahzmg antibodies In suinmaiv this analysis has identified two entical regions ot the toxm Λ piotem auamst which neutiahzmg antibodies aie pioduced as assayed bv the ( HO neutiahzation assav
EXAMPLE 13
Production And I valuation Ol Avian Antitoxin Against ( difficile Recombinant loxin A Polypeptide
In I xample 12 we demonstrated neutralization oi toxm \ mediated cvtotoxicitv bv alf itv punlled antibodies leactive to iecombinant toxin A protem lo determine whether antibodies laised against a iecombinant polypeptide fragment ol ( difficile loxin A mav be elfective m ti eating elostndial diseases antibodies to iecombinant toxin A piotem representing the binding domain were generated Two toxin A binding domain recombinant polypeptides expressing the binding domain in either the pMAI c (pMAI 870-2680) oi pLT 23(pPA1870- 2680) vector were used as immunogens The pM l piotein was affinity purified as a soluble product |1 xample 12(d)] and the pf I protein was isolated as insoluble inclusion bodies [Example 12(d)] and solubihzed to an immunologicallv active protein using a proprietary method desciibed in a pending patent application (US Patent Application Senal No 08/129027) I his I xample involves (a) immunization (b) antitoxin collection (c) determination ot antitoxin antibodv met. (d) anti-tecombmant toxm A neutiahzation of toxin A hemaLiilutinution activity in
Figure imgf000088_0001
and (e) assay ot in vino toxm A neutiuliznm activity a) Immunization
T he soluble and the inclusion body preparations each were used separately to immunize hens. Both purified toxin A polypeptides were diluted in PBS and emulsified with approximately equal volumes of CFA for the initial immunization or IFA for subsequent booster immunizations. On day zero, for each of the recombinant preparations, two egg lay ing w hite Leghorn hens (obtained from local breeder) were each iiηected at multiple sites ( intramuscular and subcutaneous) w ith 1 ml of recombinant adjuv ant mixture containing approximately 0.5 to 1 .5 mgs of recombinant toxm A Booster immunizations ol" 1 .0 mg were giv en on day s 14 and day 28
10 b) Antitoxin Collection l otai y olk immune IgY was extracted as described in the standard PT G protocol (as in 1 xample 1 ) and the final IgY pellet w as dissolv ed in sterile PBS at the original y olk v olume I his material is designated "immune recombinant luY" or "immune I LΪY "
I s c) Antitoxin Antibody Titer l o determine if" the recombinant toxm A protein was sufficiently immunogenic to raise antibodies in hens, the antibody titer of a recombinant toxin A polypeptide w as determined by ELISA E gs from both hens were collected on day 32. the yolks pooled and the antibody 0 was isolated using PEG as described, fhe immune recombinant IgY antibody liter was determined lor the soluble recombinant protem containing the maltose binding protein fusion generated in p-Mal ( pMA I 870-2680) Ninety-six well falcon Pro-bind plates w ere coated overnight at 4°C w ith 100 μl /well ol toxin A recombinant at 2.5 μg /μl in PBS containing 0 05% thimerosal Another plate w as also coated with maltose binding protem ( MBP ) at the
25 same concentration, to permit comparison ot antibody reactivity to the fusion partner The next day . the wells were blocked w ith PBS containing 1 % bovine serum albumin (BSA) for 1 hour at 37°C. IgY isolated from immune or preimmune eggs was diluted in antibody diluent ( PBS containing ! % BSA and 0.05% fween-20). and added to the blocked wells and incubated for 1 hour at 37°C . The plates were washed three times w ith PBS w ith 0.05%
30 f ween-20. then three times w ith PBS Alkaline phosphatase conjugated rabbit anti-chicken
IgG (Sigma) diluted 1 : 1000 in antibody diluent was added to the plate, and incubated for 1 hour at 37°C. The plates were washed as before and substrate was added, [p-nitrophenyl phosphate ( Sιgma)| at 1 mg/ml in 0 05M Na 'O,. pH 9 5 and 10 mM MgCL. The plates were evaluated quantitatively on a Dynatech MR 300 Micro EPA plate reader at 410 nm about 10 minutes after the addition of substrate.
Based on these ELISA results, high antibody titers were raised in chickens immunized with the toxin A recombinant polypeptide. The recombinant appeared to be highly immunogenic. as it was able lo generate high antibody titers relatively quickly with few immunizations. Immune IgY titer directed specifically to the toxin A portion of the recombinant was higher than the immune IgY titer to its fusion partner, the maltose binding protein, and significantly higher than the preimmune IgY. ELISA titers ( reciprocal of the highest dilution of IgY generating a signal) in the preimmune IgY to the MBP or the recombinant was < 1 :30 while the immune IgY titers to MBP and the toxin A recombinant were 1 : 1 8750 and > 1 :93750 respectively. Importantly, the anti-reeombinant antibody titers generated in the hens against the recombinant polypeptide is much higher, compared to antibodies to that region raised using native toxin A. 1 he recombinant antibody titer to region 1 70-2680 in the CT A antibody preparation is at least five-fold lower compared to the recombinant generated antibodies ( 1 : 18750 versus 1 :93750). T hus, it appears a better- immune response can be generated against a specific recombinant using that recombinant as the immunogen compared to the native toxin A.
This observation is significant, as it shows that because recombinant portions stimulate the production of antibodies, it is not necessary to use native toxin molecules to produce antitoxin preparations. T hus, the problems associated with the toxicity of the native toxin are avoided and large-scale antitoxin production is facilitated.
d) Anti-Rccombinant Toxin A Neutralization Of Toxin A
Hemagglutination Activity In Vitro T oxin A has hemagglutinating activity besides cytotoxic and enterotoxin properties.
Specifically, toxin A agglutinates rabbit erythrocytes by binding to a trisaccharide ( gal 1 -3131 - 4GlcNAc) on the cell surface. [H. Krivan et al.. Infect. Immun.. 53 :573-581 ( 1986). ] W e examined whether the anti-recombinant toxin A ( immune IgY. antibodies raised against the insoluble product expressed in pET) can neutralize the hemagglutination activity of toxm A in vitro, fhe hemagglutination assay procedure used was described by I EC. Krivan el al.
Polyethylene glycol-fractionated immune or preimmune IgY were pre-absorbed w ith citrated rabbit erythrocytes prior to performing the hemagglutination assay because we have found that IgY alone can agglutinate red blood cells. Citrated rabbit red blood cells (RRBC\s)(Coeaiico) were washed twice by centrifugation at 450 x g with isotomc buffer (01 M Tπs-HCI.005 M NaCl pH 72) RRBC-ieactive antibodies in the IgY were removed bv preparing a 10% RRBC suspension (made bv adding packed cells to immune oi preimmune IgY) and incubating the mixture toi 1 houi at 37°C The RRBCs weie then removed bv centrifugation s Neutiahzation of the hemagglutination activity of toxin A bv antibodv was tested in lound- bottomed 96-well mictotiter plates Iwentv-flve μl ot toxm A (36 μg /ml) (Tech Lab) in isoto c butter was mixed with an equal volume of different dilutions ol immune or preimmune I ^ in isoto c butfei and incubated for 15 minutes al orn temperatuie I hen. 50 μl ol ,ι I' o RRBC suspension in isotomc buftei was added and the mixtuie was incubated 0 loi houis at 4°( Positive contiol wells containing the final concentration ol 9 μg/ml of toxm \ alter dilution without IgY were also included Hemagglutination activity was assessed visually with a diffuse matrix of RRBC s coating the bottom ot the well lepresentinL! a positive hemauglutination reaction and a tight button of RRBC s at the bottom ot the well representing a negative icaction Ihe anti-recombinant immune ig\ neutralized s toxin \ hemauglutmalion activity giving a neutralization liter ol 18 However preimmune
[g\ was unable lo neutralize the hemagglutination ability ol toxin A
e) \ssa\ Ol //; Vttio loxin A Neutralizing AetiMtv
Ihe abihtv ol the anli-recombinant toxin A IgY (immune IgY antibodies uused against 0 p\l \I870-2680 the soluble iecombinant binding domain piotein expressed in pι\l \L designated as Anti-tox \-2 in 1 iguie 14 and referred to as iecombinant icgion 6) and pie- immune \ι\ piepaied as descπbed in E ample 8(c) above to neutralize the cvtotoxic activity ol toxin
Figure imgf000091_0001
using the CHO cell cvtotoxicitv assav and toxm A ( lech 1 ub) at a eoncentiation ol 0 lug ml as described m Example 8(d) above \s additional s controls the anti-native toxm \ Ig (CTA) and pre-immune IgY prepaiations described in
I xample 8(e) above were also tested Ihe results aie shown in Tiguie 14
The unti-iecombinanl toxin \ IgY demonstrated only paitial neutiahzation of the cvtotoxic activity ot toxin A while the pre-immune IgY did not demonstrate anv significant neulraiizinLi activity EXAMPLE 14
In
Figure imgf000092_0001
Neutralization Of ( difficile loxin A
The ability oi avian antibodies (IgY) raised against iecombinant toxm A binding domain to neuttahze the enterotoxin activity oi ( difficile toxin A was evaluated in vivo using Golden Svnan hamsters The Example involved (a) preparation of the avian anti- leeombinant toxin A IgY for otal administration (b) in \ι\o protection of hamsteis trom ( difficile toxm A entcrotoxicitv bv treatment ot toxm A with avian anti-reeombinant toxm A Is_Y and (e) histolomc evaluation of hamster ceca
a) Preparation Of The Avian Anti-Recombinant Toxin A Ig\ For Oral Administration
Lgμs were collected liom hens which had been immunized with the recombinant ( dilficile toxin A tiagment pMA1870-2680 (described in Example 13 above) \ second gioup ol eggs putchused at a local supermarket was used as a pre-immune (negative) control I g volk immunoglobulin (IgY) was extiacted bv PEG liom the two gioups ol eggs as described in I xample 8(c) and the final Ig' pellets were solubihzed in one-fourth the onginal volk volume using 0 I cuibonate butler (mixture ot NalICO
Figure imgf000092_0002
Na ( O ) pll 9 s Ihe basic carbonate butter was used m oidei to protect the toxin A liom the acidic pll ol the stomach environment
b) /// vivo Protection Of Hamsters Against C. difficile I
Figure imgf000092_0003
A Entcrotoxicitv By Treatment Of Toxm A With Avian Anti- recombinant Toxin A IgY In older to assess the ability ol the avian anti-reeombinant toxm A IgY piepaied in section (a) above to neutralize the in vno enteiotoxin activity ot toxin A an in
Figure imgf000092_0004
toxin neutiahzation model was developed using Golden Svnan hamsters I his model was based on published values toi the minimum amount ot toxin A lequired to elicit diairhea (008 mg toxin A kg bodv vvt ) and death (016 mg toxin A kg bodv wt ) in hamsteis when administered oiallv (I veilv a al Infect Immun .47349-3^2 (1985)
I or the studv loin separate experimental gioups were used with each gioup consisting ol 7 lemule Golden Svnan hamsters (Charles Rivei) approx thiee and one-hall weeks old weighing approx 50 gms each The animals were housed as groups of 3 and 4. and were offered tood and water ad libitum through the entire length of the studv
I or each animal, a mixtuie containing either lOμg ot toxm A (02 mg/Kg) or 30μg of toxin A (06 mg/kg) (( difficile toxin A was obtained from lech Lab and 1 ml of either the anti-iecombinant toxin A IgY or pre-immune IgY (Irom section (a) above) was prepared These mixtuies were incubated at 37°C lor 60 nun and were then administered to the animals bv ihe oial route The animals were then observed lor the onset ot diarrhea and death toi a period ol 24 hrs following the administration ol the toxin * IgY mixtuies at the end of which time the follow mg results were tabulated and shown in I able 17
IABLΓ 17
Studv Outcome At 24 Hours
Figure imgf000093_0002
Vmmals icmained htalthv thiough the entire 24 hour studv peiiotl Animals developed diaiihca but did not die Animals developed diarrhea and subsequently died
Ptetieatment ol toxin A at both doses tested, using the anti-iecomb ant toxin A Ig\ pi evented all overt svmptoms ol disease in hamsteis Thereloie pietreatment ol ( difficile toxin A using the anti-iecombinant toxin A IgY. neutralized the in
Figure imgf000093_0001
enterotoxin activity ol the toxin \ In contiast all animals tiom the two groups which received toxin A which ^ had been pietieated using pre-immune IgY developed disease symptoms which ranged Irom diarrhea to death Ihe diairhea which developed in the 5 animals which did not die in each ot the two pre-immune groups, spontaneously resolved bv the end of the 24 hr studv period
c) Histologic Evaluation Of Hamster Ceca 0 In older to further assess the ability of anti-reeombinant toxin A IgY to protect hamsters tiom the enteiotoxin activity ol toxin A. histologic evaluations were performed on the ceca ol hamsters fiom the studv described in section (b) above
Ihiee gioups ol animals weie sacrificed in order to piepare histological specimens I e first gioup consisted of a single representative animal taken fiom each of the 4 gioups ol
- 9! surviving hamsters at the conclusion ot the studv described in section (b) above These animals represented the 24 hr timepoint of the studv
The second group consisted ot two animals which were not part of the studv described above but were separately treated with the same toxin A t pre-immune IgY mixtures as desciibed lor the animals in section (b) above Both ot these hamsters developed diarrhea, and were sacrificed 8 hrs after the time of admmistiation ot the toxin A - pie-immune IgY mixtuies \t the time ol sacrifice both animals were presenting symptoms ol diarrhea These animals icpresented the acute phase ol the studv
Ihe ϊma] group consisted ot a single untreated hamster tiom the same shipment ol animals as those used lor the two previous groups I his animal served as the normal control
Samples ot cecal tissue were removed from die 7 animals described above and were fixed overnight at 4°C using 10% buttered formalin the fixed tissues were paraffin- embedded sectioned and mounted on glass microscope slides ihe tissue sections were then stained using hematoxv hn and eosrn (II and E stain) and were examined bv light microscopy Ihe tissues obtained liom the two 24 hi animals which leceived mixtuies containing either l μg oi 3()μg ol toxin A and anti-iecombinant toxin A lg weie indistinguishable from the noimal control both in terms ol gross pathology as well as at the microscopic level I hese observations piovidc further evidence for the ability ol anti-iecombinant toxin A IgY to etlectivelv neutiahze the in
Figure imgf000094_0001
and thus its ability to prevent acute oi lasting toxin A-induced pathology
In contrast the tissues Irom the two 24 hr animals which received the toxm \ + preimmune I<Λ mixtuies demonstrated significant palhologv In both ol these moups the mucosal lavei was observed to be less oiganized than in the noimal control tissue Ihe cytoplasm ol the epithelial cells had a vacuolated appeatanee and gaps weie piesent between the epithelium and the underlying cell layers The lamina pi pπa was laigelv absent
Intestinal vilh and crypts were significantly diminished and appealed to have been overgrown bv a planai laver ot epithelial cells and llbroblasts Iheretoie although these animals ovcrtlv appealed to lecover tiom the acute symptoms ot toxm A intoxication lasting pathologic alterations to the cecal mucosa had occurred Ihe tissues obtained liom the two acute animals which received mixtuies of tox A and pie-immune IgY demonstrated the most significant pathology \l the gross pathological level both animals eie observed to have severely distended ceca which were filled with watery diairhea-hke material \t the microscopic level, the animal that was given the mixture containing l Oμg of toxin A and pre-immune IgY was found to have a mucosal layer w hich had a ragged, damaged appearance, and a disorganized, compacted quality. The crypts were largely absent, and numerous breaks in the epithelium had occurred. There was also an influx of erythrocytes into spaces between the epithelial layer and the underlying tissue. The animal w hich had received the mixture containing 30μg of toxin A and pre-immune IgY demonstrated the most severe pathology. The cecal tissue of this animal had an appearance v ery similar to that observed in animals which had died from C. difficile disease. Widespread destruction of the mucosa was noted, and the epithelial layer had sloughed. I Iemorrhagic areas containing large numbers of erythrocytes were very prevalent. All semblance of normal tissue architecture was absent from this specimen. In terms of the presentation of pathologic events, this in vivo hamster model oϊ toxin A-intoxication correlates v ery closely with the pathologic consequences of C. difficile disease in hamsters. The results presented in this Example demonstrate that while anti-reeombinant toxin A ( Interv al 6) IgY is capable of only partially neutralizing the cytotoxic activ ity of C. difficile toxin A. the same antibody effectiv ely neutralizes 100% of the in vivo enterotoxin activilv of the toxin. While it is not intended that this invention be limited to this mechanism, this may be due to the eytotoxicity and enteroioxicity of ( '. difficile 'l oxin A as two separate and distinct biological functions.
EXAMPLE 15 In l ivo Neutralization Of C. Difficile T oxin A By-
Antibodies Against Recombinant Toxin A Poly peptides
T he ability of avian antibodies directed against the recombinant ( '. difficile toxin A fragment 1 70-2680 (as expressed by pMA 1870-2680: see E.xample 13 ) to neutralize the enteroto.xic activity of toxin A was demonstrated in E.xample 14. T he ability of avian antibodies ( IgYs) directed against other recombinant toxin A epitopes to neutralize native to in A in vivo was next ev aluated. This example involved: (a) the preparation of" IgYs against recombinant toxin A poly peptides: (b) in vivo protection of hamsters against toxin A by treatment with anti-reeombinant toxin A IgYs and (c) quantification of specific antibody concentration in CTA and Interval 6 IgY PEG preparations.
T he nucleotide sequence of the coding region of the entire toxin A protein is listed in SEQ I D NO:5. The amino acid sequence of the entire toxin A protein is listed in SEQ II) O:6. T he amino acid sequence consisting of amino acid residues 1 870 through 2680 of toxin A is listed in SEQ ID NO 7 The amino acid sequence consisting of am o acid lesidues 1870 thiough 1960 ot toxin A is listed in SFQ ID NO 8
a) Preparation Of IgY's Against Recombinant I oxin A Polypeptides
I ggs were collected trom 1 eghorn hens which have been immunized with recombinant ( difficile loxin \ polvpcptide tiagments encompassing the entire toxm A protem fhe polypeptide tiagments used as immunogens were 1) pMA 1870-2680 (Inteival 6) 2) pPA 1100-1450 (Inteival 4) and 3) a mixture of fragments consisting ot p 30-300 (Inteival 1)
10 pMA 300-660 (Interval 2) pMA 660-1100 (Interval 3) and pMA 1450-1870 (Interval 5)
I his mixtuie ot immunogens is leteπed to as Interval 1235 | location ot each inteival within the toxin A molecule is shown in 1 igure 15 \ I n I rgure 1 A the following abbiev unions uic used pP reteis to the ρl f23 vector (New I nuland BioLabs) pM rclcrs to the pMAI , '-c vectoi (New 1 ngland Biolabs) A lelers to toxin A the numbers refer lo the
1 amino acid inteival expressed in the clone (Tor example the designation pM \30-300 indicates that the iecombinant clone encodes amino acids 30-100 oi toxin \ and tlie vector used was p AI I -e)
Ihe iecombinant pioteins were generated as desciibed in I xample II Ihe lι_\s were extiacted and solubihzed in 01 M caibonate butter pll 95 f i oial administration as described
20 in I xample 14(a) Ihe IgY teactivities against each individual recombinant interval was evaluated bv I 1 ISA as desciibed in I xample 13(e)
b) //; Vtvo Protection Ol Hamsters Against Toxin
A Bv Treatment With Anti-Rccombinant Toxin A Antibodies
-> The ability ot antibodies raised against recombinant toxin A polvpeptides to provide m piotection against the enterotoxic activity ot toxin A was examined in the hamster model system 1 his assav was pertormed as described Example 14(b) Bπcllv loi each 40-50 giam female Golden Svnan hamster (Charles River) 1 ml ol ILI\ 4X (; t resuspended in 1/4 ol the oπuinal volk volume) PI G prep against Interval 6 Interval 4 or Interval 1235 was 30 mixed wilh 30 μg (1 DMI[) oiai dose) of ( difficile toxin A ( lech I ab) Pieimmune IgY mixed with toxin A served as a negative control Antibodies laised against ( difficile toxoid \ (f xample 8) mixed with toxin A (C A) served as a positive contiol Ihe mixtuie was
1 n1eubatcd loi I houi at 37°C then orallv admmisteied to lightly etherized hamsteis using an I8G feeding needle fhe animals were then observed tor the onset ot diarrhea and death for a period of approximatelv 24 houis The results are shown in Table 18
TABLΓ IS
Studv Outcome Alter 24 Hours
Figure imgf000097_0002
shows no smn ot illness developed diarrhea but did not die Animal developed diatrhea and died
Pie tieatment ol toxin A with Ig s against Interval 6 prevented diarrhea in 6 ot 7 hamste s and completely pi evented death in all 7 In contrast as with pieimmunc Igi Ig s against Interval 4 and Interval 1235 had no effect on the onset ol diarrhea and death m the hamsiei s
c) Quantification Of Specific Antibodv ncentration In ( I \
\nd Interval 6 IgY PEG Preparations lo determine the puπtv ot IgY I G preparations
Figure imgf000097_0001
I 870-2680 (Interval 6) l< PI G pieparation was chromatographed using IIPI C and a KW-803 sizing column (Shodcx) ihe resulting piofile ot absorbance at 280 nm is shown in I igure 16 Ihe single laige peak coiiesponds to the piedicted MVv ol Ig") Integration ol the area undei the single laige peak showed that greater than 95% of the piotein eluted liom the column was piesent in this single peak This result demonstrated that the majoiitv ( 95%) ot the material absoibing at 280 nm in the PEG preparation corresponds to IgY I herefore absorbance at 280 nm can be used to determine the total antibodv concentration m PEG preparations l determine the concentration ol Interval 6-speeiflc antibodies (expiessed as percent ol total antibodv) within the CTA and pMA1870-2680 (Interval 6) PEG ptepuiations defined quantities ol these antibodv preparations were affinity punfied on a pPAl 870-2680(11) (shown schematically in i iguie I5B) affinity column and the specific antibodies were quantified In 1 iguie 15B the following abbreviations aic used pP reteis to the pi 123 veetoi (New 1 ngland Biol abs). pM icfers to the pMAI IM-e vector (New 1 ngland BioLubs). pG refers to the pGEX vector (Pharmacia): pB refers to the PinPointIM Xa vector (Promega): A refers to toxin A; the numbers refer to the amino acid interval expressed in the clone. Ihe solid black ovals represent the MBP: the hatched ovals represent glutathione S-transfcrase: the hatched circles represent the biotin tag: and ]]\U\ represents the poly-histidine tag. An affinity column containing recombinant toxin A icpeat protein was made as follows 1 our ml ol" PBS-washed Λctigel resin (Sterogene) was coupled with 5-10 mg of pPΛ 1870-2680 inclusion body protein |prepared as described in Example (17) and dialyzed into PBS| in a 15 ml tube (falcon) containing 1/10 final volume Λld-couphng solution (1 M sodium eyanoborohydride) Aliquots of the supernatant from the coupling reactions, betore and after coupling, were assessed by Coomassie staining of 75% SDS-PAGE. gels. Based upon protein band intensities, greater than 6 mg ot recombinant protein was coupled to the resin. Ihe resin was poured into a 10 ml column (BioRad). washed extensively with PBS. pre-eluted with 4 guanidine-HCI ( 10 mM Iπs-HCl. pll 80.0005" J. thimerosal) and re- equihbrated with PBS Ihe column was stored at 4°C. Aliquots ot a pMAI 870-2680 (Interval 6) or a CTA IgY polycional antibody preparation (PEG prep) were alfinity purified on the above alllnilv column as follows Ihe column was attached to an I'V mom toi (ISCO) and washed with PBS I or pMA 1870-2680 IgY purification, a 2X PEG prep (filter sterilized using a 045 μ filter: approximately 500 mg total IgY) was applied. The column was washed with PBS until the baseline was re- established (ihe column flow-through was saved), washed with BBS 1 ween to chile nonspeeifically binding antibodies and re-equilibrated with PBS Bound antibody was eluted Irom the column in 4 M guanιdιnc-11C1 (in 10 mM lπs-IICl. pll 80.0005% thimerosal) Ihe entire elution peak was collected m a 15 ml tube (I alcon) Ihe column was re- equilibraled and the column eluate was le-chromalographed as described above Ihe antibody preparation was quantified by I'V absorbance (the elution buffer was used to zero the spectrophotometer) Total purified antibody was approximately 9 mg and 1 mg from the first and second chromatography passes, respectively. Ihe low yield trom tlie second pass indicated that most specific antibodies were removed by the first round ol chromatography The estimated percentage of Interval 6 specific antibodies in the p A 1870-2680 PEG prep is approximately 2%
I he percentage of Interval 6 specific antibodies in the CIA PEG prep was determined (utilizing the same column and methodology described above) to be approximately 05% ot total ILLY A 4X PEG prep contains approximately 20 mg/ml IgY. Thus in b) above, approximately 400 μg specific antibody in the Interval 6 PEG prep neutralized 30 μg toxin A
5 EXAMPLE 16
//; Vivo treatment Of C difficile Disease In Hamsters By Recombinant Interval 6 Antibodies
fhe ability of antibodies directed against recombinant Interval 6 ot toxin A to protect 10 hamsters in vivo from (' difficile disease was examined this e.xample involved (a) prophv lactic treatment oi C difficile disease and (b) therapeutic treatment oϊ C difficile disease
a) Prophylactic Treatment Of difficile Di.sease
I ^ this experiment was perlormed as described in E.xample 9(b) Three groups each consisting ol 7 female 100 gram Syrian hamsters (Charles River) weie prophv laeticallv treated with either preimmune IgYs. IgYs against native toxm A and B [CIΛB. see E.xample 8 (a) and (b)| oi IgYs against Interval 6 IgYs were prepared as 4X PEG preparations as described in Example 9(a)
-0 Ihe animals were orally dosed 3 times daily, roughly at 4 houi intervals, loi 12 days with 1 ml antibody preparations diluted Ensure K Using estimates of specific antibody concentration Irom Example 15(e). each dose of the Inteival 6 antibody prep contained approximately 400 μg of specific antibodv On day 2 each hamster was predisposed to ( difficile infection by the oial administration of 30 mg ol" CTιndamycm-1 IC1 (Sigma) in I ml
25 ol water On day 3 the hamsters were orally challenged with I nil of (' difficile inoculum strain ATCC" 43596 in sterile saline containing approximately 100 organisms Ihe animals were then observed for the onset of" diarrhea and subsequent death dunng the treatment period Ihe results are shown in Table 19 TABLE 1')
I cthalitv Alter 12 Davs 01 Ircatment
Figure imgf000100_0001
heatment ot hamsters with orallv-administered IgYs against Interval 6 successfully protected 7 out ot 7 (100%) ol the animals from ( difficile disease One ol the hamsters m this group presented with diarrhea which subsequently resolved during the course ot tieatment \s shown pieviouslv in Example 9 antibodies to native toxin A and toxin B weie highly pioteetive In this 1 xample 6 out of 7 animals suivived in the C 1 AB treatment group Ml ot the hamsters tieated with pieimmunc sera came down with diarihea and died the suivivots in both the T \B and Interval 6 groups remained healthv throughout a 12 dav post- treatment period In particular () out of 7 Interval 6-trcatcd hamsters suivived at least 2 weeks after termination ot treatment which suggests that these antibodies provide a long- lasting euie I hese icsults icpresent the first demonstration that antibodies generated against a iecombinant lemon ol toxin \ can prevent CDΛD when administered passively to animals 1 hese icsults also indicate that antibodies laised against Interval 6 alone mav be sulllcient to pioteet animals Irom ( difficile disease when administered piophv laeticallv
Pieviouslv others had raised antibodies against toxm \ bv actively immunizing hamsters against a recombinant polypeptide located within the Interval 6 region |I verlv DM a al (1990) Cuir Miciobiol 2129] 1 igure I 7 shows schematically the location of the 1 verlv el al tiu-lnteiv l 6 iecombinant protein (cloned into the pUC veetoi) in compuiison with the complete Interval 6 eonstiuct (pMA I 870-2680) used herein lo generate neutralizing antibodies duected against toxin A In I igure 17 the solid black oval represents the MBP which is fused to the toxin A Interval 6 in pMAI 870-2680
Ihe Lvcrlv cl al antibodies (mtru-Intcrval 6) were onlv able to paitiallv pioteet hamsters against ( difficile infection in terms ot survival (4 out ol 8 animals suivived) and luithetmoie these antibodies did not prevent diairhea in anv oi the animals Additionally animals tieated with the intia-lntcrv l 6 antibodies [I verlv a al (1990) supia] died when tieatment was lcrnoved
In conliast the experiment shown above demonstrates that passive admmistiation ol anti-Interval 6 antibodies pievented diarrhea in 6 out ol 7 animals and completely pievented death due to CDAD. furthermore, as discussed above, passive administration of the anti- Interval 6 antibodies provides a long lasting cure (i e . treatment could be withdrawn without incident)
b) Therapeutic Treatment Of C. difficile Disease: /// Vivo
Treatment Of An Established C. difficile Infection In Hamsters With Recombinant Interval 6 Antibodies
The ability ot antibodies against recombinant interval 6 of toxin A lo theiapeutically Heat C difficile disease was examined fhe experiment was perfoimed essentially as descπbed in E.xample 10(b) Three groups, each containing seven to eight female Golden
Syrian hamsters ( 100 g each. Charles River) were treated with either preimmune IgY, IgYs against native toxin A and toxin B (CfAB) and IgYs against Interval 6 Ihe antibodies were piepaied as described above as 4X PEG preparations
Ihe hamsters were first predisposed to ( difficile infection with a 3 mg dose ol Chndamycin-HCT (Sigma) administered orally in 1 ml of water Approximately 24 hrs later, the animals were orally challenged with 1 ml of C difficile strain A fCC 43596 m sterile saline containing approximately 200 organisms. One day after infection, the presence of toxin A and B was determined in the feces ot the hamsters using a commercial immunoassay kit (Cytoelone A-B EPA. Cambridge Biotech) to verify establishment of infection I our members of each group were randomly selected and tested. Teces from an unmfected hamster was tested as a negative control. All infected animals tested positive for the presence of toxin accoiding to the manufacturer's procedure The initiation of treatment then started approximately 24 hi post-infection
Ihe animals were dosed daily at roughly 4 hr intervals with 1 mi antibody preparation diluted in Ensure K (ROSS Labs) fhe amount of specific antibodies given per dose
(determined by affinity purification) was estimated to be about 400 μg of anti-Interval 6 IgY (for animals in the Interval 6 group) and 100 μg and 70 μg of anti-toxin A (Interval 6- speciflc) and anti-tox B (Interval 3-speciflc: see Example 19). respectively, for the CTAB preparation Ihe animals were treated for 9 days and then observed foi an additional 4 days lor the presence of diarrhea and death The results indicating the number ol survivors and the number of dead 4 days post-infection are shown in fable 20 TABLC 20
In M\<> therapeutic Treatment With Interval 6 Antibodies
Figure imgf000102_0001
directed against both Interval 6 and CTAB successfully pievented death liom ( difficile when thcrapeuticailv administered 24 hr after infection I his result is significant since manv mvestigatois begin therapeutic treatment ot hamsters with existing chugs (e g vancomycin. phenellamvcns tiacumicrns etc ) 8 hr post-mteciion [Swanson et al ( 1991 ) Antimicrobial Agents and Chemotherapy 351108 and ( 1989) I Antibiotics 42941 I oitv-two percent ol hamsters tieated with preimmune lg\ died liom C DAD While the anti-Interval 6 antibodies prevented death in the treated hamsters thev did not eliminate all symptoms ol C DAD as 3 animals presented with slight diarrhea In addition, one C I AB- liealed and one pieimmune-treated animal also had diarrhea 14 davs post-mfeetion I hese icsults indicate that anti-Inteival 6 antibodies provide an effective means ot therapy loi CDΛD
EXAMPLE 17
Induction Ol loxin A Neutiahzmg Antibodies Requnes Soluble Interval 6 Protein
\s shown in I xamplcs 11(d) and 15 expression ot iecombinant pioteins in / coli mav lesult in the production ol either soluble oi msoluble piote II msoluble piotem is pioduced the recombinant protein is solubihzed pnor to immunization ol animals lo deteimme whether, one or both of the soluble or insoluble iecombinant proteins could be used to generate neutralizing antibodies to loxin A. the tollovving experiment was perfoimed This example involved a) expression ol the toxin A icpeats and sublragments ot these icpeats in / coli using a vancty ot expression vectors b) identification ot iecombinant toxm A repeats and sub-regions to which neutiahzmg antibodies bind and c) determination oi the neutiahzation ability ol antibodies laised aua st soluble and insoluble toxin A icpeat immunogen a) Expression Of The Toxin A Repeats And Subfragments Of
These Repeats In E. coli Using A Variety Of Expression Vectors
The Interval 6 immunogen utilized in Examples 15 and 16 was the pMA 1870-2680 piotem. which the toxin A repeats aic expressed as a soluble fusion protein with the MBP
(described in Example 11) Interestingly, expression ot this region (trom the SpcΛ site to the nd ol the repeats see f igure 15B) in three other expiession constiucts. as either native (pPΛ I 870-2680). polv -I (is tagged fpPΛ 1870-2680 (II)] or biotin-tagged (pBA 1870-2680) pioteins lesultcd in completely insoluble protem upon induction ol the baetenal host (see 1 iguie I 5B) The host strain BI 21 (Novagen) was used for expression of pBA 1870-2680 and host stum B( 21(Dr 3) (Novagen) was used foi expiession of pPA1870-2680 and pPΛI870- 2680(11) I hese insoluble pioteins accumulated to high levels in inclusion bodies t xpression ol iecombinant plasmids in / eoli host cells giown in 2X \ medium was perfoimed as desciibed |Wιlhams et al (1995). sup/a] \s summanzed in Tigure 15B expression of fragments ot the toxin \ repeats (as either Vtcrminul Spel-I coR] tiagments. or C -terminal I eoR\-end fragments) also yielded high levels oi insoluble piotein using pGLX (pGAl 870-2190) PιnPoιnt'"-Xa (pBA 1870-2190 and pB \225()-2680) and pi f expiession systems (pPA1870-2190) Ihe pGI X and pE I expression systems are described in 1 xample 11 Ihe PmPoιntI -Xa expression system drives the expiession ol lusion pioteins in L coli I usion pioteins fiom PιιιPoιnt' -λu vectors contain a biotm tag at the ammo-terminal end and can be affinity punfied Soft! ink"" Soft Release avi in lesin (Piomega) under mild denaturing conditions (5 mM biot )
Ihe solubility ol expiessed piole ns Irom the pPG1870-2190 and pP \1870-2190 expiession constiucts was determined alter induction ol iecombinant protein expiession undei conditions lepoited to enhance piotem solubility [These conditions comprise gio th ol the host at reduced temperature (3()°C) and the utilization ot high (1 mVl 1PIG) oi low (01 mM 1PTG) concentrations ol mducer [Williams el al (1995). supia] \ll expressed iecombinant toxin A piotein was insoluble under these conditions Thus, expression ot (hese tiagments of the toxin A lepeats in pLI and pCEX expression vectors icsults in the production ot insoluble leconibinunt protein even when the host cells are grown at ieduced temperatuie and using lower concentrations ol the lndueer Although expression of these fragments in pMal vectois yielded affinity punflable soluble lusion protein the protein was either predominantly insoluble (pMA 1870-2190) or unstable (pMA2250-2650) \ttcrnpts to solubihze expressed protein from the pMA 1870-2190 expression construct using reduced temperature or lower inducer concentration (as described above) did not improve fusion protein solubility.
Collectively, these results demonstrate that expression of the loxin A repeat region in E. coli results in the production of msoluble recombinant protein, when expressed as either large ( aa 1 870-2680) or small (aa 1870-2190 or aa 2250-2680) fragments, in a v ariety oϊ expression vectors (native or pol -his tagged pET. pGEX or PinPoint I M- Xa vectors), utilizing growth conditions shown to enhance protein solubility . The exception to this rule were fusions w ith the MBP. which enhanced protein solubility, either partially (pMA 1 870-2190) or fully ( pMA 1 870-2680).
b) Identification Of Recombinant Toxin A Repeats And Sub-
Regions To Which Neutralizing Antibodies Bind
Toxin A repeat regions to w hich neutralizing antibodies bind were identi fied bv utilizing recombinant toxin A repeat region proteins expressed as soluble or insoluble proteins to deplete protectiv e antibodies from a polycional pool of antibodies against native ( '. difficile toxin A. .An in vivo assay was developed to evaluate proteins for the ability to bind neutralizing antibodies. fhe rational for this assay is as follows. Recombinant proteins were first pre-mixed w ith antibodies against native toxin A (CTA antibody : generated in Example 8 ) and allowed lo react. Subsequently. ( '. difficile toxin A was added at a concentration lethal to hamsters and the mixture was administered to hamsters via I P injection. I f the recombinant protein contains neutralizing epitopes. the CTA antibodies would lose their ability to bind toxin A l esulting in diarrhea and/or death of the hamsters.
T he assay was performed as follows. T he lethal dose of toxin A when delivered orally to nine 40 to 50 g Golden Syrian hamsters (Sasco) was determined to be 10 to 30 μg. T he
PEG-puriflcd CTA antibodv preparation was diluted to 0.5X concentration ( i. e.. the antibodies were diluted at twice the original yolk volume) in 0. 1 M carbonate buffer. pH 9.5. fhe antibodies were diluted in carbonate buffer to protect them from acid degradation in the stomach. T he concentration of 0.5X was u.sed because it was found to be the lowest effective concentration against toxin A. T he concentration of Interval 6-specifie antibodies in the 0.5X
CT A prep w as estimated to be 10- 1 5 μg/ml (estimated using the method described in Example 1 5 ). The inclusion body preparation ( insoluble Interval 6 protein: pPA l 870-2680(11)] and the soluble Interval 6 protein [pMA 1870-2680: see figure 15] were both compared for their ability to bind to neutralizing antibodies against C. difficile toxin A (CTA). Specifically. 1 to 2 mg of recombinant protein was mixed with 5 ml of a 0.5X CTA antibody prep (estimated lo contain 60-70 μg of Interval 6-specillc antibody ). After incubation for 1 hr at 37°C. CTA ( Tech Lab) al a final concentration of 30 μg/ml was added and incubated for another I hr at 37°C. One ml of this mixture containing 30 μg of toxin A (and 10- 15 μg of Interval 6- speei tlc antibodv) was administered orallv to 40-50 g Golden Syrian hamsters ( Sasco). Recombinant proteins that result in the loss of neutralizing capacity of the CTA antibodv would indicate that those proteins contain neutralizing epitopes. Preimmune and CTA antibodies ( both at 0.5X ) without the addition of any recombinant protein served as negative and positive controls, respectively.
I wo other inclusion body preparations, both expressed as insoluble products in the pE'f v ector, were tested: on containing a different insert (toxin B fragment ) other than Interv al 6 cal led pPB 1 50-2070 (see figure 1 8) which serves as a control for insoluble I nterv al 6. the other w as a truncated v ersion of the Interval 6 region called pPΛ I 870-2 1 90 ( see figure I 5B ). fhe results of this experiment are shown in fable 2 1 .
TABLE 21
Binding Ol Neutralizinu A ntibodies Bv Soluble interval 6 Protein Studv Outcome A lter 24 Hours
Figure imgf000105_0001
C difficile lox in A (CTA ) was added to each group. An imals showed no signs ot" i l lness. A nimals developed diarrhea but did not die. Animals developed diarrhea and died.
Preimmune antibody was ineffective against toxin A. while anti-C f A antibodies at a dilute 0.5X concentration almost completely protected the hamsters against the enterotoxic effects ol" CTA. The addition of recombinant proteins pPB 1 850-2070 or pPA 1 870-2 1 0 to the anti-CTA antibody had no effect upon its protective ability, indicating that these recombinant proteins do not bind to neutralizing antibodies. On the other hand, recombinant Interval 6 protein was able to bind to neutralizing anti-CTA antibodies and neutralized the in pioteetive effect of the anti-CTA antibodies I oui out ol live animals in the group tieated with anti-CTA antibodies mixed with soluble Interval 6 piotein exhibited toxin a ssociated toxicity (diarrhea and death) Moreover the results showed that Interval 6 proicin m list be expressed as a soluble product in older lor it to bind to neutiahzmg anti-C I A antibodies since the addition ot insoluble Interval 6 protem had no effect on the neutralizing capacity ol the C I A antibodv piep
e) Determination Of Neutralization Ability Ot Antibodies Raised Against Soluble And Insoluble loxin A Repeat
Immunogen lo determine it neutralizing antibodies are induced against solubihzed inclusion bodies msoluble toxin \ repeat protein was solubihzed and specific antibodies were raised in chickens Insoluble pPΛI 870-2680 piotem was solubihzed usinu the method described in V» ilhams ι / al (1995) supra Briefly induced cultures ( 500 nil) were pelleted bv ecntiilugation at 3000 X g for 10 mm at 4°( [he cell pellets were icsuspended thoroughly m 10 ml of inclusion bodv sonication buffei (25 mM HI PES pll 77 100 mM kC 1 125 mM MgCI 20% glveerol 01% (v/v) onidet P-40 1 M DTI ) The suspension was tiansfeired to a 30 ml non-glass centrifuge tube I ive bundled μl ol 1 nm/ml lv ozvme was added and the tubes were incubated on ice lor 30 mm Ihe suspension was then frozen at -70°( loi at least I hi Ihe suspension was thawed rapidly in a water bath at loom temperature and then placed on ice Ihe suspension was then sonicated using at least eight Is see bursts ot the mieroprobe (Bianson So calor Model No 450) with intermittent cooling on ice
Ihe sonicated suspension was tianslerred to a 35 ml Oakndge tube and eeniπli ed at 6000 X g lor 10 mm at 4°C to pellet the inclusion bodies Ihe pellet was washed 2 times bv pipetting oi voitexing in iiesh. ice-cold RIPA butler |01% SDS 1% Tiiton λ-100 1% sodium deowcholate m TBS (2s mM Ins-Cl pll 75 15() mM NaC 1)| Ihe inclusion bodies were leeentπtuged alter each wash Ihe inclusion bodies were dπed and tianslerred using a small metal spatula to a 15 ml tube (I aleon) One ml of 10% SDS was added and the pellet was solubihzed bv gentlv pipetting the solution up and down using a 1 l mieropipettoi Ihe solubihzation was facilitated bv heating the sample to 9s°C when neeessaiv
Once the inclusion bodies were in solution the samples were diluted with 9 volumes ot PBS Ihe protem solutions were dialvzed overnight against a 100-fold volume ol PBS containing 005% SDS at loom temperature The dialvsis buffer was then changed to PBS containing 001% SDS and the samples were dialyzed for several hours to overnight at room tempeiature The samples were stored at 4°C until used Pπoi to further use. the samples were w imed to loom temperature to allow anv piecipitatcd SDS to go back into solution
Ihe inclusion body preparation was used to immunize hens fhe protem was diaivzed into PBS and emulsified with approximately equal volumes ot CTA lor the initial immunization oi If Λ lot subsequent booster immunizations On dav zeio lor each of the iecombinant iecombinant preparations, two egg laving white I eghorn hens were each injected at multiple sites (IM and SC) with 1 ml of recombinant proieiπ-udμivant mixtuie containing approximatelv 05-1 5 mg ol recombinant protein Booster immunizations of 10 mg were given ol davs 14 and dav 28 I ggs were collected on dav 32 and the antibodv isolated using PI G as desciibed in I xample 14(a) High titers ol toxin \ specific antibodies were present (as assayed bv 1 I 1S\ using the method described in f xample 13) liters were determined lot both antibodies against iecombinant polypeptides pPA 1870-2680 and pMA I 870-2680 and were lound to be comparable at I 62500
\ntibodies against soluble Inteival 6 (pMAI 870-2680) and insoluble Interval 6 |(ιικlusιon bodv) pPA1870-2680] were tested tor neutralizing ability against toxin A using the //; assav desciibed in 1 xample 15(b) Pieimmunc antibodies and antibodies against toxin \ (C I \) served as negative and positive controls respectively Ihe icsults aie shown
TABLF 22 cuti.ili/.ition Ol loxin A Bv Antibodies Auainst Soluble Interval 6 Piotein Studv Outcome Λltci 24 Houis
Figure imgf000107_0002
Animals showed no sign of illness Λnimal developed diaπhea but did not die Λnimal developed dianiiea and died
Antibodies raised against native loxin A were protective while preimmune antibodies had little etlect As found using the in CHO assay [described in f xample 8(d)) where antibodies laised against the soluble Interval 6 could paitiallv neutiahze the etfects ol toxin A here thev were able to completely neutiahze toxin A in
Figure imgf000107_0001
In contrast, the antibodies raised against the insoluble Interval 6 was unable to neutralize the effects of toxin A in vivo as shown above (Table 22) and in vitro as shown in the CHO assay [described in Example 8(d)].
These results demonstrate that soluble toxin A repeat immunogen is necessary to induce the production ot neutralizing antibodies in chickens, and that the generation of such soluble immunogen is obtained only with a specific expression vector ( pMal) containing the toxin A region spanning aa 1870-2680 T hat is to say. insoluble protein that is subsequently solubihzed does not result in a toxm A antigen that w ill elicit a neutralizing antibodv
EXAMPLE 18
Cloning And Expression Of The ( difficile T oxin B Gene
f he toxin B gene has been cloned and sequenced: the ammo acid sequence deduced ti om the cloned nucleotide sequence predicts a MW of 269.7 k lor toxm B | Barroso et al . \ucT. Acids Res 1 8:4004 ( 1990)]. I he nucleotide sequence ot the coding region of the entire toxin B gene is listed in SEQ I D NO. The amino acid sequence ol the eniite toxin B protein is l isted in SEQ ID NOT 0 I he ammo acid sequence consisting ol ammo acid residues 1 850 through 2360 ot toxin B is listed in SEQ I D NO 1 1 I he ammo acid sequence consisting ol amino acid residues 1 750 through 2360 ot toxin B is l isted in SI Q ID NO- 12 Giv en the expense and difficulty of isolating native toxin B protein, il would be adv antageous lo use simple and inexpensiv e procaryotic expression sy stems lo produce and purify high lev els ol recombinant toxin B protein lor immunization purposes Ideally , the isolated recombinant protem would be soluble in ol der to preserv e nativ e anligenicity . ince solubihzed inclusion bodv proteins often do not fold into nativ e eonlormations Indeed as show n in Example 1 7. neutralizing antibodies against recombinant toxin A wer e only obtained w hen soluble recombinant toxin A polypeptides were used as the immunogen. l o allow ease ol puri fication, the recombinant protein should be expressed to lev els gι eater than 1 mg/hter of E coli culture. fo determine w hether high lev els of recombinant toxin B protein could be produced in I. c oh. ti agments of the toxin B gene were cloned into various prokary otic expression v ectors, and assessed for the ability to express recombinant toxin B protein in E coli I his f xample inv olv ed (a) cloning of the toxin B gene and (b) expression of the toxin B gene in /: co . a) Cloning Of The Toxin B Gene
The toxin B gene was cloned using PCR amplification from C. difficile genomic DNA. Initially, the gene was cloned in two overlapping fragments, using primer pairs P5/P6 and P7/P8. The location of" these primers along the toxin B gene is shown schematically in figure 18. The sequence of each of these primers is: P5: 5" TAGAAAAAATGGCΛΛΛTGT 3" (SEQ
ID NO:l I ): P6: 5" TTTCΛTCTTGTA GAGTCAAAG 3" (SEQ ID NO: 12): P7: 5" GATGCCΛCAΛGATGATTTΛGTG 3* (SEQ ID NO: I 3): and P8: 5" CTAATTGAGCTGTATCAGGATC 3' (SEQ ID NO: 14). figure 18 also shows the location of the following primers along the loxin B gene: P9 which consists of the sequence 5' CGGAATTCCTAGAAAAAATGGCAΛ ATG 3" (SEQ ID
NO: 15): P10 which consists of the sequence 5" GCTCTAGAΛTGA CCΛTAAGCTAGCCΛ 5" (SEQ ID NO: 16): PI 1 which consists of the sequence
5" OGGAΛTTCGAGTTGGTΛGAΛΛGGTGGA .3" (SEQ ID NO: 17): PI 3 which consists of the sequence 5" CGGAΛ I fCGGT TΛTTATCTTΛAGGATG 3' (SEQ ID NO: 18): and PI4 which consists of the sequence 5" CGGΛATTCTTGATAACTGGAT TTGTGAC 3" (SEQ ID
NO: 1 ). fhe amino acid sequence consisting of amino acid residues 1852 through 2362 of toxin B is listed in SEQ ID NO:20. The amino acid sequence consisting of amino acid residues 1755 through 2362 of toxin B is listed in SEQ ID NO:21.
Clostridium difficile VPI strain 10463 was obtained from the American Type Culture Collection (ATCC 43255) and grown under anaerobic conditions in brain-heart infusion medium (Becton Dickinson). High molecuiar-weight ('. difficile DNA was isolated essentially as described [Wren and Tabaqchaii (1987) J. Clin. Microbiol..25:2402). except I) 100 μg/ml proteinase k in 0.5% SDS was used to disrupt the bacteria and 2) cetytrimethvTammonium bromide (CTAB) precipitation jas described by Ausubel et al. Eds.. Current Protocols m Molecular Biologw Vol.2 (1989) Current Protocols) was used to remove carbohydrates from the cleared lysate. Briefly, after disruption of the bacteria with proteinase k and SDS. the solution is adjusted to approximately 0.7 M NaCl by the addition of a 1/7 volume of 5M Nad. A 1/10 volume of C'f AB/NaCl (10% CTAB in 0.7 M NaCl) solution was added and the solution was mixed thoroughly and incubated 10 min at 65°C. An equal volume of chloroform/isoamyT alcohol (24:1) was added and the phases were thoroughly mixed. The organic and aqueous phases were separated by centrifugation in a microfuge for 5 min. The aqueous supernatant was removed and extracted with phenol/chloroform/ isoamyl alcohol (25:24:1 ). The phases were separated by centrifugation in a microfuge for 5 min. The supernatant was transferred to a fiesh tube and the DNA was precipitated with isopropanol The DNA precipitate was pelleted bv bnef centrifugation in a microfuge The DNA pellet was washed with 70% ethanol to remove residual CIΛB Ihe DNA pellet was then dried and ledissolved in TI buffei (10 mM Ins-HCl pH80 I mM I DT\) Ihe integiitv and yield ol genomic DNA was assessed bv comparison with a serial dilution ot uncut lambda
DNA alter electrophoresis on an agarose gel loxin B fiagments were cloned bv PCR utilizing a proolieading thermostable DNA polvmeiuse |natιve Pfu polvmerase (Stratagene)j Ihe high lldehtv of this polvmeiase leduees the mutation problems associated with amplification bv eiroi ptone polvmerases (e g I acf polvmerase) PCR amplification was performed using the PC R primer pans P5 (SI Q ID
NO I 1) with P6 (SI Q ID NO 12) and P7 (SI Q ID NO 13) with P8 (SI Q ID NO 14) in 5Q μl leactions containing 10 mM Tiis-HCl pH83 50 mM KC I 1 "s mM MgCI 200 μM ol each cIN IP 02 μM each primer and 50 ng ( difficile ueiiomie DN v Reactions weie overlaid with 100 μl mineral oil heated to 94°C loi 4 mm 0 sμl native Pfu polvmerase (Stratagene) was added and the reactions were cycled 30 times at 94°( lor 1 nun 5()°C toi 1 m 72°C (2 nun loi each kb ot sequence lo be amplified) followed bv 10 mm at 72°C Duplicate teactions were pooled chloiotorm extracted and ethanol piecipitated After washing in 70% ethanol the pellets were resuspended in 5() \.ι\ II butler (10 mM Ins-HCl pH80 1 mM fDIA) Ihe P5/P6 amplification product was cloned into pU( 19 as outlined below 1 Oj.il aliquots ol DNΛ were gel purified using the Prep-a-Gene kit (BioRad) and Imated to ma\ lestiicted pDC 19 vccloi Recombinant clones were isolated and confirmed bv testnction digestion usinu standaid iecombinant molecular biologv techniques (Sambiook (/ al 1989) Inserts liom two independent isolates were identified in which the toxm B insert was onented such that the vector Bamϊ and Sac] sites were 5 and 3 onented lespectivelv (pi CB10-
1530) Ihe mscrt-conlain g Bam ]I acl Iragment was cloned into simtluilv cut pi I23a-e veetoi DNA and piotein expression was induced in small scale cultuies (5 ml) ot identified clones
I otal piotein extracts were isolated lesolved on SDS-PAGE gels and toxin B piote identified bv Western analysis utilizing a goat anti-toxin B altinitv punfied antibodv (Tech
I ab) Piocedures lor protem induction SDS-PAGE and Western blot analysis were performed as described m Williams a al (1995) supia In bnef " ml cultuies ol bacteria giown in 2XYT containing 100 μg/ml ampiciilin containing the uppiopπute iecombinant clone were induced to express recombinant protein by addition of IPTG to ImM. The E. coh hosts u.sed were: BE2KDE3) or BL21(DE3)LysS (Novagen) for pET plasmids.
Cultures were induced by the addition of IPTG to a final concentration of 1.0 M when the cell density reached 05 ODfi()0, and induced protein was allowed to accumulate for two hrs after induction Prote samples were prepared by pelleting 1 ml ahquots of bacteria by centrifugation (I nun in microfuge). and resuspension of the pelleted bacteria in 150 μl of 2X SDS-PAGE sample buffer (0.125 mM Tris-HCI pH 68.2 mM EDTA.6% SDS.20% glycerol.0025% bromυphenol blue: β-mercaptoethanol is added to 5% before use). The samples were heated to 95°C for 5 nun. then cooled and 5 or 10 μls loaded on 75% SDS- PAGE gels High molecular weight protein markers (BioRad) were also loaded, to allow estimation ol the MW of identified fusion proteins. After electrophoresis. protem was detected either generally by staining the gels with Coomassie Blue, or specifically, by blotting to nurocellulose loi Western blot detection of specific immunoreactive protein Ihe MW of induced toxin B icaetive piotem allowed the integrity ot the toxm B reading frame to be determined
Ihe pL I 23b recombinant (pPBIO-1530) expressed high MW iecombinant toxm B icaetive protein, consistent with predicted values This confirmed that trame terminating errors had not occurred during PCR amplification A pET23b expression clone containing the l()-1750aa interval of the toxin B gene was constructed, by fusion of the EcoRV-Spe] Iragment ol the P7/P8 amplification product to the P5-EcoRV interval ot the P5/P6 amplification product (see 1 igure 18) in pPBlO-1530 fhe integrity ol this clone (pPBK)- 1750) was confirmed by restriction mapping, and Western blot detection ol expressed recombinant toxm B prote fevels of induced protein liom both pPB10-153() and pPBlO- 1750 weie too low ιo facilitate purification ol usable amounts ol iecombinant toxin B protein fhe remaining 1750-2360 aa interval was directly cloned into pMAE or pET expression vectors trom the P7/P8 amplification product as described below
b) Expression Of The Toxin B (iene i) Oven icvv Of Expression Methodologies I ragments of the toxin B gene were expressed as either native or lusion proteins in E coli Native proteins were expressed in either the pl_r23a-c or pETI6b expression vectors (Novagen) fhe pET23 vectors contain an extensive polylinker sequence in all three reading frames (a-c vectors) followed by a C-terminal poly-histidine repeal. The pET16b vector contains a N-terminal poly-histidine sequence immediately 5' to a small polylinker. The poly-histidine sequence binds to Ni-Chelate columns and allows affinity purification of tagged target proteins [Williams el al (1995), supra]. I hese affinity tags arc small (10 aa for pET16b.6 aa for pET23) allowing the expression and affinity purification ol native proteins with only limited amounts ol foreign sequences
An N-terminal lustidine-tagged derivative of pE I 16b containing an extensive cloning cassette was constructed to facilitate cloning of N-terminal poly-histidine tagged toxin B expressing constructs This was accomplished by replacement of the promoter region oi the pl'T23u and b vectors with that of" the pET16b expression vector Each vector was restricted with BglU and h'de\. and the reactions resolved on a 1.2 % agarose gel The pET 16b promoter region (contained in a 200 bp BglU-Nde] fragment) and the promoter-less pET23 a or b vectors were cut trom the gel. mixed and Prep-A-Gene (BioRad) purified fhe eluted DNA was ligated. and transformants screened for promoter replacement by \co\ digestion ol purified plasmid DNA (the pET16b promoter contains this site, the pEf23 promoter does not) I hese clones (denoted pETHisa or b) contain the pE I 16b promoter (consisting ol a p I 7-lac piomoter. ribosome binding site and poly -histidine (lOaa) sequence) fused at the A'del site to the extensive p I23 polylinker
All MBP lusion proteins were constructed and expressed in the pMAI IM-c or pMAI I -p2 vectors (New England Biolabs) in which the protem of interest is expressed as a C-iermmal lusion with MBP All pET plasmids were expressed in either the BI 21(DE3) or
BE21( f 3)l.ysS expression hosts, while pMal plasmids were expressed in the BI 21 host
1 aige scale (500 mis to I liter) cultures ot each recombinant were giovvn in 2X Y I broth, induced, and soluble piotein fractions were isolated as described [Williams, et al (1995). supra] The soluble protem extracts were affinity chromatographed to isolate recombinant fusion protein, as described [Williams et al . (1995) supra] In brief, extracts containing tagged pET fusions were chromatographed on a nickel chelate column, and eluted using imidazoie salts or low pll (pll 40) as described by the distributor (Novagen oi Qiagen) 1 xtracts containing soluble pMAI lusion protein were prepared and chromatogiaphed in PBS buffet over an amylose resin (New England Biolabs) column, and eluted with PBS containing 10 mM maltose as described [Williams et al (1995). supia] ii) Overview Of Toxin B Expression
In both large expression constructs described in (a) above, only low level ( i. e.. less than 1 mg/litcr of intact or nondegraded recombinant protein) expression of" recombinant protein was detected. A number of expression constructs containing smaller fragments of the toxin B gene were then constructed, to determine if small regions of the gene can be expressed to high levels { i. e.. greater than 1 mg/liter intact protein) without extensive protein degradation. All were constructed by in frame fusions of convenient toxin B restriction fragments to either the pMAL-e. pET23a-c. pETT 6b or pETHisa-b expression vectors, or by engineering restriction sites at specific locations using PCR amplification [using the same conditions described in (a) above). In all cases, clones were verified by restriction mapping, and. w here indicated. DNA sequencing.
Protein preparations from induced cultures of each of these constructs were analyzed, by SDS-PAGE. to estimate protein stability (Coomassie Blue staining) and immunoreactivity against anti-toxin B specific antiserum ( Western analysis). Higher levels of intact ( i. e. . nondegraded). full length fusion proteins were observed with the smaller constructs as compared with the larger recombinants. and a series of expression constructs spanning the entire toxin B gene were constructed ( figures 1 8. 19 and 20 and T able 23).
Constructs that expressed significant levels of recombinant toxin B protein (greater than I mg/liter intact recombinant protein) in E. coli were identified and a series of these clones that spans the toxin B gene are shown in figure 1 9 and summarized in T able 23.
T hese clones were utilized to isolate pure toxin B recombinant protein from the entire to m B gene. Significant protein y ields w ere obtained from pMAE expression constructs spanning the entire toxin B gene, and yields oϊ full length soluble fusion protein ranged from an estimated I mg/liter culture (pMB l 100- 1530) to greater than 20 mg/liter culture (pMB 1750-2360). Representative purifications of MBP and poiy-histidine-tagged toxin B recombinants are show n in figures 21 and 22. f igure 21 shows a Coomassie Blue stained 7.5% SDS- PAGE gel on which v arious protein samples extracted from bacteria harboring pMB I 50- 2360 were electrophoresed. Samples were loaded as follows: Eane 1 : protein extracted from uninduced culture: Eane 2: induced culture protein: Eane 3 : total protein from induced culture after sonication: Lane 4: soluble protein: and Lane 5: eluted affinity purified protein, figure
22 depicts the purification of recombinant proteins expressed in bacteria harboring either pPB 1850-2360 (Lanes 1 -3) or pPBl 750-2360 (Lanes 4-6). Samples were loaded as follows: uninduced total protein ( Lanes I and 4): induced total protein ( fanes 2 and 5): and eluted affinity purified protem ( Lanes 3 and 6) The broad range molecular weight protein markers (BioRad) are shown in Lane 7
Thus, although high level expression was not attained using large expression constructs trom the toxin B gene, usable lev els of recombinant protem were obtained by isolating induced protein from a series ot smaller pMAL expression consti ucts that span the entire toxm B gene
These results represent the first demonstration ol the f easibility oi expressing recombinant toxin B protem to high lev els in L coli Λs well, expression ol small legions ol the putativ e ligand binding domain (repeat region) ol toxin B as native protein yielded insoluble protein, while large constructs, or f usions to MBP were soluble (f iguie 19), demonstrating that specific methodologies are necessary to produce soluble tusion protein tiom this interval
iii) Clone Construction And Expression Details \ poi tion of the toxin B gene containing the toxin B lepeat region | amιno acid residues 1 852-2362 ol toxin B (SEQ ID NO 20)| was isolated bv PCR amplification of this interv al ot the toxm B gene Irom C ' diffic ile genomic DNA I he sequence, and location w ithin the toxin B gene, of the tw o PCR primers [ P7 ( SFQ ID NO 1 3 ) and P8 ( SEQ ID NO 14) | used to amplify this region are shown in f igure 18 DNA li om the PCR amplification was purified by chlorof orm extraction and ethanol piec pitation as described above fhe DNA was icstπcted w ith Spe\ and the cleav ed DNA w as icsolv ed by agarose gel electrophoresis I he icstriction digestion w ith S/xJ cleav ed the 3 6 kb amplification product into a 1 8 kb doublet band l lus doublet band was cut Irom the gel and mixed with appropriately cut. gel purified pMALc or pFT23b vector I hese v ectors were prepared by digestion w ith IlindWl. filling in the over hanging ends using the Klenow enzyme, and cleaving with Xbal ( pMALc) or Nhel ( pET23b) The gel purified DNA liagments were purified using the Prep-A-Gene kit ( BioRad ) and the DNA was ligated. transtoi med and putative recombinant clones analvzed by restriction mapping pi 1 and pMal clones containing the toxin B lepeat inser t (e\a interv al 1 750-2360 ol toxm B) were verified by restriction mapping, using enzvmes that cleaved specific sites within the toxin B icgion In both cases fusion ot the toxin B .S/;el site w ith either the compatible Yha site (pMal) or compatible \ hel site (pET) is predicted to create an in frame tusion I his was confirmed in the case of the pMB l 750-2360 clone by DNA sequencing ot the clone junction and 5' end of the toxin B insert using a MBP specific primer (New England Biolabs). In the case of the pET construct, the fusion of the blunt ended toxin B 3 " end to the filled Hindlll site should create an in-frame fusion with the C-terminal poly-histidine sequence in this vector. The pPB 1750-2360 clone selected had lost, as predicted, the Hindlll site at this clone junction: this eliminated the possibility that an additional adenosine residue was added to the 3' end of the PCR product by a terminal transferase activity of the Pfu polymerase. since fusion of this adenosine residue to the filled Hindlll site would regenerate the restriction site (and was observed in several clones).
One liter cultures of each expression construct were grown, and fusion protein purified by affinity chromatography on either an amylose resin column (pMAL constructs: resin supplied by New England Biolabs) or Ni-chelatc column (pET constructs: resin supplied by Qiagen or Novagen ) as described [ Williams et al. ( 1995 ). supra]. The integrity and purity of the fusion proteins w ere determined by Coomassie staining oϊ SDS-PAGE protein gels as well as Western blot analysis with either an affinity purified goat polycional antiserum (Tech Lab). or a chicken polycional PEG prep, raised against the toxin B protein ( CTB) as described above in { .xample 8. In both cases, affinity purification resulted in yields in excess of 20 mg protein per liter culture, of which greater than 90% was estimated to be full-length recombinant protein. I t should be noted that the poly-histidine affinity tagged protein was released from the Qiagen Ni-NTA resin at low imidazoie concentration (60 mM ). necessitating the use ol" a 40 mM imidazoie rather than a 60 mM imidazoie wash step during purification.
A periplasmically secreted v ersion of pMB l 750-2360 was constructed by replacement of the promoter and MBP coding region of this construct with that from a related vector ( pMAI. I N'-p2; New England Biolabs) in which a signal sequence is present at the N-terminus of the MBP. such that fusion protein is exported. This was accomplished by substituting a
Bgll -EcoRM promoter fragment from pMAL-p2 into pMB l 750-2360. The yields of secreted, affinity purified protein ( recovered from osmotic shock extracts as described by Riggs in Current Protocols in Molecular Biology. Vol. 2. Ausubel. el al.. Eds. ( 1989). Current Protocols, pp. 16.6. 1 - 16.6. 14] from this vector (pMBp! 750-2360) were 6.5 mg/liter culture. of which 50% was estimated to be full-length lusion protein. fhe interval was also expressed in two non-overlapping fragments. pMB 1 750- 1970 was constructed by introduction of a frameshift into pMB l 750-2360. by restriction with Hindl ll . filling in the overhanging ends and rciigation of the plasmid. Recombinant clones were selected by loss ot the Hindlll site, and further restriction map analysis Recombinant protem expression trom this vector was more than 20 mg/hter ol greater than 90% pure protein
The complementary region was expressed in pMB 1970-2360 fins construct was created bv icmoval ot the 1750-1970 interval of pMB I 750-2360 I his was accomplished by lestrietion ol this plasmid with EcoRl (in the pMalc polylinker 5 to the insert) and III. filling in the overhanging ends, and rehgation ol the plasmid The resultant plasmid. pMB 1970-2360. was made using both intracellularly and secreted versions ol the pMBl 750-2360 vector No fusion protein was secreted in the pMBp 1970-2360 veision. perhaps due to a contoimational constraint that prevents export of the fusion protem However, the intracellularly expressed vector produced greater than 40nιg/hter of greater than 90% full- length lusion protein
C onstructs to precisely express the toxin B repeats in either pMalc (pMBl 850-2360) oi pi I 16b (pP I 850-2360) were constructed as follows The DNA interval including the toxm B icpeats was PCR amplified as described above utilizing PCR primers P14 (S1Q ID NO 19) and P8 (SEQ ID NO 14) Pnmer P14 adds a LcoRl site immediately flanking the stall ot the toxin B icpeats
The amplified Iragment was cloned into the pi 7 Blue I -vector (Novagen) and recombinant clones in which single copies ot the PCR tiagment were inserted in either onentation were selected (p I 71850-2360) and confirmed by restiiction mapping Ihe insert was excised tiom two appropriately oriented independently isolated pi 71850-2360 plasmids. with EcoRl (5" end ot lepeats) and PstI (in the flanking polvhnkei ot the vector), and cloned into / coRli Psil cleaved pMalc veetoi The resulting construct (pMB1850-2360) was confirmed bv lestrietion analysis, and yielded 20 ιng/1 ol soluble fusion protein [greater than 90% intact (/ e . nondegraded)] after affinity chromatography Restriction oi this plasmid with
Hindlll and ieligation of the vector tesulted in the removal ot the 1970-2360 interval The lesultant construct (pMBl 850- 1970) expressed greater than 70 mg/hter ot 90% lull length fusion piotem The pPB 1 850-2360 construct was made by cloning a EcoRl (filled with Klenow)- Ba l fragment from a pT71850-2360 vector (opposite orientation to that used in the pMB 1850-2360 construction) into Ndel (filledVZΪαwHI cleaved pETlόb vector. Yields of" affinity purified soluble fusion protein were 15 mg/liter. of greater than 90% full length fusion protein.
Several smaller expression constructs from the 1750-2070 interval were also constructed in His-tagged pET vectors, but expression of these plasmids in the BL21 (DE3) host resulted in the production of high levels of mostly insoluble protein (see Table 23 and figure 19). These constructs were made as follows. pPB I 850- l 970 was constructed by cloning a Bg/ll-Hindlll fragment of" pPB l 850-2360 into Bg/ll/Hindlll cleaved pET23b vector. pPB l 850-2070 was constructed by cloning a BglU-Pvull fragment of pPB l 850-2360 into Bglll/Hincl l cleaved pET23b vector. pPB 1 750- 1970(c ) was constructed by removal of the internal Hindlll fragment of a pPB 1 750-2360 v ector in which the vector Hindlll site was regenerated during cloning (presumably by the addition of an A residue to the amplified PCR product by terminal transferase activity oϊ Pfu polvmerase). "fhe pPB l 750- 1970(n) construct was made by insertion of the insert containing the iX e - Hindlll fragment of pPB l 750-2360 into identically cleaved pETHisb v ector. All constructs were confirmed by restriction digestion.
An expression construct that directs expression of the 10-470 aa interval of toxin B was constructed in the pMalc vector as follows. A Nhe (a site 5" to the insert in the pET23 vector )-A fl] 1 (filled) fragment of" the toxin B gene from pPB 10- 1530 was cloned into Xbal ( compatible w ith \'hel )l Hindlll (filled) pMalc vector. T he integrity of the construct ( pMB I O- 470) was verified by restriction mapping and DNA sequencing of the 5' clone junction using a MBP specific DNA primer ( Ne England Biolabs). However, all expressed protein was degraded to the MBP monomer MW.
A second construct spanning this interval (aa 10-470) was constructed by cloning the PCR amplification product from a reaction containing the P9 (SEQ ID NO: I 5 ) and P 10 (SEQ ID NO: 16) primers (Figure 18) into the pETHisa vector. This was accomplished by cloning the PCR product as an /:'cv;RI-blunt fragment into EcoRl-HincΛ l restricted vector DNA: recombinant clones were verified by restriction mapping. Although this construct (pPB I O-
520) allowed expression and purification (utilizing the N-terminal polyhistidine affinity tag) of intact fusion protein, yields were estimated at less than 500 μg per liter culture.
15 Higher yield of recombinant protein from this interval (aa 10-520) were obtained by expression of the interval in two overlapping clones The 10-330aa interval was cloned in both pi THisa and pMalc vectors, using the BamHl-4fllll (filled) DNA fragment liom pPBlO- 520 1 his tiagment was cloned into Bamlll-Hi dlll (filled) restricted pMalc or BamHl-Hincll restricted pLTElisa vector Recombinant clones were verified by restriction mapping High level expression ot either insoluble (pLT) or soluble (pMal) lusion protein was obtained lotal yields ol lusion protein tiom the pMB10-330 construct (ligurc 18) were 20 mg/hter culture, ot which 10%) was estimated to be lull-length fusion protein \lthough yields of this interval were higher and >90% lull-length recombinant protein produced when expiessed from the pFT construct, the pMal fusion was utilized since the expressed piotein was soluble and thus more likely to have the native conformation
The pMB260-520 clone was constructed by cloning EcoRl- Xbal cleaved amplified DNA tiom a PCR reaction containing the PI 1 (SfQ ID NO 17) and Plϋ (SEQ ID NO 16) DNA pnmers (1 igure 18) into similarly lestπctcd pMalc vector Yields ol affinity purified protem were 10 mg/hter ot which approximately 50% was estimated to be lull-length iecombinant protein
Ihe aa5I0-l 110 interval was expressed as described below 1 his entne interval was expressed as a pMal lusion bv cloning the hhel-Hmdlll fragment ot pUCBlO-1530 into Xhal- I ndlll cleaved pMalc vector Ihe integrity of the construct (pMB510-l 110) was verified by lestrietion mapping and DNA sequencing of the 5 clone junction using a MBP specific DNA primer Ihe vield ol affinity purified protem was 25 mg/hter cultuie ol which 5% was estimated to be lull-length lusion protein (1 mg/hte ) lo attempt to obtain higher yields, this region was expressed two tiagments (aa510- 820 and 820-1110) in the pMalc vector 1 he pMB510-820 clone was constiucted by inset tion ol a Sad (in the pMalc pol linker 5" to the insert )-Hpal DNA Iragment from pMB510-l 110 into Sad/Slul restncied pMalc vector Ihe pMB820-l 110 vector was constiucted by inseition ol the Hpal-llindll fragment ot pi IC BI 0- 1530 into BamHI (filled) Hindlll cleaved pMalc vector Ihe integrity ot these constructs were venfied by restriction mapping and DNA sequencing ol the 5 clone junction using a MBP specific DNA primer Recombinant protem expressed tiom the pMB510-820 vector was highly unstable
However, high levels (20 mg/hter) ot 90% full-length tusion piotein were obtained tiom the pMB820-l 105 construct Ihe combination of partially degraded pMB510-l 110 piotein (enriched for the 510-820 interval) with the pMB820-l 110 protein provides usable amounts of recombinant antigen from this interval
The aal 100-1750 interval was expressed as described below The entire interval was expressed in the pMalc vector from a construct in which the tT(fiIled)- /?t'I fragment of pPB10-1750 was inserted into Slul/Xhal (Xbal is compatible with Spel. Siul and filled Accl sites are both blunt ended) restricted pMalc The integrity of this construct (pMBl 100-1750) was verified by restriction mapping and DNA sequencing of the clone junction using a MBP specific DNA primet Although 15 mg/hter of affinity purified protein was isolated from cells haibonng this construct, the protem was greater than 99% degraded to MBP monomer size A smaller derivative of pMBl 100-1750 was constructed by restriction of pMBl 100-
1750 with ,4/711 and Sail (in the pMalc polylinker 3' to the insert), filling in the overhanging ends, and lehgaimg the plasmid fhe resultant clone (verified bv restriction digestion and DNA sequencing) has deleted the a l 530- 1750 interval, was designated pMBl 100-1530 pMBl 100-1530 expiessed recombinant protem al a yield of greater than 40 mg/hter. of which 5% was estimated lo be full-length fusion piotem
I hree constructs were made to express the remaining interval Initially, a BspHl (fllied)-.S/;el fragment fiom pPBlO-1750 was cloned into £
Figure imgf000119_0001
cleaved pMalc veetoi Ihe integrity ol this construct (pMB1570-l 750) was verified by restriction mapping and DNA sequencing of the 5" clone junction using a MBP specific DNA primer 1 xpression ol iecombinant protein from this plasmid was very low. approximately 3 mg affinity purified protein per liter, and most was degraded to MBP monomer size This region was subsequently expressed trom a PCR amplified DNA fragment \ PCR reaction utilizing primers PI 3 [SEQ ID NO 18. P13 was engineered to introduce an EcoRl site 5 to amplified toxm B sequences] and P8 (SEQ ID NO' 14) was performed on (' difficile genomic DNA as described above The amplified fragment was cleaved with EcoRl and Spel. and cloned into
EcoRllXhal cleaved pMalc veetoi The resultant clone (pMB1530-l 750) was verified by lestrietion map analysis, and recombinant protein was expressed and punfied Ihe yield was greater than 20 mg protem per liter culture and it was estimated that 25% was full-length tusion protein, this was a significantly higher yield than the original pMB I 570-1750 clone fhe inseit of pMB 1530- 1750 (in a EcoRl-Sall fragment) was transferred to the pETHisa vector (EcoRllXhol cleaved. Xhol and Sail ends are compatible) No detectable fusion protein was purified on Ni-Chelate columns from soluble lysates of cells induced to express lusion protein trom this construct
17 TABLE 23
Figure imgf000120_0001
C lones in italics aie clones cm ieiitly utilized to purity recombinant pi otem liom each selected interval oceurs with the CTB antibody-recombinant mixture, that recombinant contains only weak or non-neutralizing epitopes of toxin B. This assay was performed as follows
Antibodies against CTB were generated in egg laying Leghorn hens as described in Example 8 The lethal dosage ( LD ,„„) of C difficile toxin B when delivered I.P into 40g female (Jolden Syrian hamsters (Charles River) was determined to be 2.5 to 5 μg. Antibodies generated against CTB and purified by PEG precipitation could completely protect the hamsters at the I P dosage determined above The minimal amount of CTB antibody needed to af ford good protection against 5 μg of CTB when injected I P into hamsters was also determined ( I X PEG prep). These experiments defined the parameters needed to test whether a given recombinant protein could deplete protective C I B antibodies.
I e cloned legions tested for neutralizing ability cov er the entire toxin B gene and were designated as Intervals (INT) 1 through 5 (see f igure 19) Approximatelv equivalent final concentrations of each recombinant polypeptide eie tested The follow ing recombinant polypeptides w ere used 1 ) a mixture of intervals 1 and 2 (IN1 - I . 2). 2 ) a mixture ol Intervals 4 and 5 (1NT-4. 5 ) and 3) Interval 3 (INI -3) Recombinant proteins (each at about I mg tolal protein) were first preincubatcd with a final C I B antibody concentration of I X [ i e . pellet dissolved original v olk v olume as described in Example 1 (c)] in a final v olume of 5 is loi I hour at 37°C vventy-fiv e μg of CTB (at a concentration of 5 μg/ml ). enough CTB to kill 5 hamsters, was then added and the mixture was then incubated toi 1 hour at 7°C fiv e. 40g female hamsters (Charles River) in each treatment group weie then each given 1 ml of the mixture I.P. using a tuberculin syringe w ith a 27 gauge needle T he results of this experiment are shown in I able 24
TABLE 24
Bindinu Of Neutrali/ina Antibodies Bv IN I 3 Protein
"7 ,
Figure imgf000121_0001
30
1 ( difficile tovin B (CTB) was added lo each group
As shown in T able 24. the addition of recombinant proteins from IN T- 1. 2 or INT-4, 5 had no effect on the m vivo protective ability of the CTB antibody preparation compared to
- 120 - EXAMPLE 19
Identification. Purification And Induction Of Neutralizing Antibodies Against Recombinant C difficile Toxin B Protein
l determine vv hethei recombinant toxin B poly peptide fragments can generate neutralizing antibodies. Iv picalh animals would first be immunized with recombinant proteins and anti-i ecombinant antibodies ate generated 1 hese anti-recombmant protein antibodies are then tested toi neuti ahzmg ability in vivo or in vin o Depending on the immunogenic nature ol the i ecombinant polypeptide. the generation of high-titer antibodies against that protein may take several months l accelerate this process and identify which recombinant polypeptide! s) may be the best candidate to generate neutralizing antibodies, depletion studies ei e pei f ormed Specifically , i ecombinant toxin B polypeptide were pre-scieened bv testing vv hethei thev hav e the ability to bind to protective antibodies trom a CTB antibodv prepai ation eind hence deplete those antibodies of their neutralizing capacity I hose recombinant polv peptides found to bind CT B, were then utilized to generate neutralizing antibodies 1 his Example involved a) identification of recombinant sub-iegions within toxin B to w hich neutralizing antibodies bind, b) identification ot toxin B sub-region specific antibodies that neutralize toxin B in vivo, and c) generation and ev aluation ot antibodies icaetiv e to i ecombinant toxin B poly peptides
a) Identification Of Recombinant Sub-Regions Within Toxin B
To Which Neutralizing Antibodies Bind
Sub-i egions w ithin toxin B to w hich neutralizing antibodies bind were identified bv utilizing i ecombinant toxin B proteins to deplete protectiv e antibodies ti om a polycional pool of antibodies against native ( ' difficile toxin B An in vivo assay was developed to evaluate protein pi eparations for the ability to bind neutralizing antibodies Recombinant proteins were first pi e-mixed w ith antibodies directed against native toxin B (CTB antibodv . see Example 8) and allowed to leact toi one hour at 37°C Subsequently . L difficile toxin B (C TB. l ech 1 ab) was added at a concentration lethal to hamsters and incubated lor another hour at 37°C f ter incubation this mixtui e was injected intraperitoneallv ( I P) into hamstei s I I the iecombinant poly peptide contains neutralizing epitopes. the CTB antibodies w ill lose its ability to protect the hamsters against death from C TB I I partial 01 complete protection
1 19 - the CTB antibody preparation alone In contrast. INT-3 recombinant polypeptide was able to remove all of the toxin B neutralizing ability of the CTB antibodies as demonstrated by the death of all the hamsters in that group
The above experiment was repeated, using two smaller expressed fragments (pMB 1 750- 1 970 and pMB 1970-2360. see Figure 19) comprising the INT- domain to determine if that domain could be fuither subdivided into smaller neutralizing epitopes. In addition, full- length INT-3 polypeptide expressed as a nickel tagged protein ( pPB l 750-2360) was tested for neutralizing ability and compared to the original INT- 3 expressed MBP fusion ( pMB I 750- 2360) f he results are shown in I able 25.
TABLE 25
Removal Ot Neutralizing Antibodies Bv Repeal Containing Proteins
Figure imgf000123_0001
( diffic ile tovin B (CT B) was added to each group
I lie i csults summarized in Table 25 indicate that the smaller polypeptide fragments within the INT-3 domain. p Bl 750- 1970 and pMB l 70-2360. partially lose the ability to bind to and remov e neutralizing antibodies from the CTB antibody pool I hese results demonstrate that the full length INT- polypeptide is required to completely deplete the CTB antibodv pool ol neutralizing antibodies This experiment also shows that the neutralization epitope ol INT-3 can be expressed in alternative vector systems and the results are independent ol the vector utilized or the accompanying fusion partner
Othei Interv al 3 specific proteins were subsequently tested for the ability to remove neutralizing antibodies within the CTB antibody pool as described abov e The Interval 3 specific proteins used in these studies are summarized in Figure 23 In Figure 23 the tollovv ing abbrev iations are used pP refers to the pET23 vector: pM refers to the pMAI c v ector: B reteis to toxin B. the numbers refer to the ammo acid interval expressed in the clone I he solid black ovals represent the MBP: and I IHH represents the poly-histidine tag Only recombinant proteins comprising the entire toxin B repeat domain (pMB 1750- 2360. pPB 1750-2360 and pPB 1850-2360) can bind and completely remove neutralizing antibodies from the CTB antibody pool. Recombinant proteins comprising only a portion of the toxin B repeat domain were not capable of completely removing neutralizing antibodies from the CTB antibody pool (pMB l 750- 1970 and pMB l 970-2360 could partially remove neutralizing antibodies while pMB 1850- 1970 and pPB 1850-2070 failed to remove any neutralizing antibodies from the C TB antibody pool ).
The above results demonstrate that only the complete ligand binding domain (repeat region ) of the toxin B gene can bind and completely remove neutralizing antibodies from the C TB antibody pool. These results demonstrate that antibodies directed against the entire toxin
B repeat region are necessary for in vivo toxin neutralization ( see Figure 23; only the recombinant proteins expressed by the pMBl 750-2360. pPB I 750-2360 and pPB 1850-2360 vectors are capable of completely removing the neutralizing antibodies from the CTB antibodv pool ). T hese results represent the first indication that the entire repeat region of toxin B would be necessary for the generation of antibodies capable of neutralizing toxin B. and that sub-regions may not be sufficient to generate maximal titers of neutralizing antibodies.
b) Identification Of Toxin B Sub-Region Specific Antibodies That Neutralize Toxin B //; Vivo
To determine i f antibodies directed against the toxin B repeat region are sufficient for neutral ization, region specific antibodies within the CTB antibodv preparation w ere affinity puri fied, and tested for in vivo neutralization. Affinity columns containing recombinant toxin B repeal proteins were made as described below . A separate affinity column was prepared using each of the follow ing recombinant toxin B repeat proteins: pPB l 750-2360. pPB 1 850- 2360. pMB 1 750- 1 970 and pMB 1970-2360.
For each affinity column to be made, four ml of PBS-washed Actigel resin ( Sterogene) was coupled overnight al room temperature with 5- 10 mg of affinity purified recombinant protein ( first extensively dialyzed into PBS) in 1 5 ml tubes ( Falcon ) containing a 1 /10 final v olume Aid-coupling solution ( 1 M sodium cyanoborohydride). Aliquots of the supernatants from the coupling reactions, before and after coupling, were assessed by Coomassie staining of 7.5% SDS-PAGE gels. Based on protein band intensities, in all cases greater than 30% coupling efficiencies were estimated. The resins were poured into 10 ml columns ( BioRad ). washed extensively with PBS. pre-eluted with 4M guanidine-HCI (in 10 M I ns-HCl. pH 8 0) and reequihbrated in PBS The columns were stored at 4°C
Aliquots of a CTB IgY polycional antibody preparation (PEG prep) were affinity purified on each of the tour columns as described below The columns were hooked to a UV ^ monitor ( I SCO). washed with PBS and 40 ml aliquots of a 2X PEG prep (filter sterilized using a 0 45 μ filter) were applied The columns were washed with PBS until the baseline v alue was re-established fhe columns were then washed w ith BBStweeπ to elute nonspeufically binding antibodies, and reequihbrated w ith PBS Bound antibody was eluted trom the column in 4M guanidine-HCI (in l OmM Tπs-HCl. pH8 0) T he eluted antibody was 0 immediately dialyzed against a 100-fold excess of PBS at 4°C for 2 hrs fhe samples were then dialv zed extensively against at least 2 changes ot PBS. and affinity purified antibody was collected and stored at 4°C T he antibody preparations wei e quantified by UV absorbance Hie elution v olumes were in the range of 4-8 ml All affinity purified stocks contained sinulai total antibodv concentrations, ranging f iom 0 25-0 35% ol the total protein applied to ^ the columns f he ability of the af Unity purified antibodv preparations to neutralize toxin B m vivo was determined using the assay outlined in a) above Affinity purified antibody was diluted 1 I in PBS betore testing The results are shown in I able 26
I n all cases similar levels of toxin neutralization was observed, such that lethality was 0 delay ed in all groups relative to preimmune controls This result demonstiates that antibodies i caetiv e to the repeat region oi the toxin B gene are suf ficient to neutiahze toxm B in \ ι\ o I he hamstei s w i ll ev entually die in all groups, but this death is maximally delay ed w ith the ( TB PI G prep antibodies I hus neutralization with the alfinity purified (AP) antibodies is not as complete as that observ ed w ith the CTB prep before af finity chromatography This 5 result mav be due to loss ot activ ity during guanidine denaturation (during the elution of the antibodies tiom the affinity column ) or the presence of antibodies specific to other regions ot the toxin B gene that can contribute to toxin neutralization ( present in the CTB PI G prep) TABLE 26
Neutralization Of l o.xin B Bv Affinity funne Antibodies
Figure imgf000126_0001
( difficile toxin B (CTB) (Tech Lab at 5 μg/ml. 25 μg total) at lethal concentration to hamstei s is added to antibody and incubated for one hour at 37V Altei incubation this mixture is miecied inirapeπtoneallv (IP) into hamsters I ach treatment group received toxin pi emixed w ith antibody raised against the indicated protein, as either 4X antibodv PEG prep ot affinity purified (AP) antibodv (trom CTB PFG pi ep. on indicated columns) Tlie amount ol specific antibody in each prep is indicated, tlie amount is direciiv detet mmed foi af f inity punfied preps and is estimated foi the 4X C I B as described in l xample 1 >
T he numbers in each uroup represent numbers ot hamsters dead oi alive 2 hr post IP administration of toxin/antibodv mixture
I he observ ation that antibodies affinity purified against the non-ov ei lapping pMB l 750- 1970 and pMB 1970-2360 proteins neutralized toxin B raised the possibility that either 1 ) antibodies specific to repeat sub-regions are sufficient to neutralize toxin B oi 2) sub-region specific proteins can bind most or all repeat specific antibodies present in the CTB polv clonal pool This would likely be due to conformational similarities between repeats, since homology in the primary amino acid sequences between dif ferent icpeats is in the range ol only 25-75% [Eichel-Streiber. et al ( 1992) Molec Gen Genetics 233 2601 I hese possibilities were tested by af finity chromatography
I he CTB PEG prep was sequentially depleted 2X on the pMB l 750- 1 70 column, only a small elution peak was observed after the second chromatography . indicating that most reactive antibodies were removed This interval depicted CTB preparation was then chromatographed on the pPB 1 850-2360 column, no antibody bound to the column I he reactiv ity ot the CTB and CTB (pMB 1 750- 1970 depleted) preps to pPB l 750-2360. pPB 1850- 2360. pMB 1 750- 1 970 and pMB l 70-2360 proteins was then determined by I L ISA using the protocol described in Example 1 (c) Briefly. 96-wcll microtitet plates ( I aleon. Pro-Bind Assay Plates) were coated with recombinant piotein by adding 100 μl volumes oi protein at 1 -2 μg/ml m PBS containing 0.005% thimerosal to each vveil and incubating ov ernight at 4°C I he next morning, the coating suspensions were decanted and the wells weie washed thiee times using PBS In order to block non-specific binding sites. 100 μl of 1 0% BSA (Sigma) in PBS (blocking solution) was then added to each well, and the plates were incubated for 1 hr at 37°C The blocking solution was decanted and duplicate samples of 150 μl of diluted antibodv was added to the fust well ot a dilution series The initial testing serum dilution vvas ( 1 /200 lot CTB prep (the concentration ot depleted CT B was standardized bv OIλ8()) in blocking solution containing 0 5% Tvveen 20. followed bv 5-fold serial dilutions into this solution This was accomplished by seually transferring 30 μl aliquots to 120 μl buffer, mixing and repeating the dilution into a fresh well Af ter the final dilution 30 ul vvas lemov ed ti om the well such that ail wells contained 120 μl final v olume A total of 5 such
10 dilutions weie performed (4 wells total) T he plates were incubated for 1 hr at 37°C f ollow ing this incubation, the senallv diluted samples weie decanted and the wells were washed three times using PBS containing 0 5% Tween 20 (PBS I ) followed bv two 5 mm washes using BBS- 1 ween and a final three washes using PBS I T o each well 100 μl ol 1 1 000 diluted secondarv antibody ( rabbit anti-chicken IgG alkaline phosphatase ( Sigma )
I ^ diluted in blocking solution containing 0 5% T ween 20] was added, and the plate was incubated 1 hr at 37°C The conjugate solutions were decanted and the plates were washed 6 times in PBS I then once in 50 mM NaX O 10 mM MgC E pH 9 5 The plates were dev eloped bv the addition ol 100 μl oi a solution containing 1 mg il paia-nitio phenv I phosphate ( Sigma) dissolv ed in 50 mM NaXO 10 mM MgC I pH9 to each well 1 he 0 plates wei e then incubated at room temperature in the dark tor -4i nun Fhe absorbencv ol each well vvas measured at 410 nm using a Dynatech MR 700 plate reader
\s pi edicted bv the alfinity chromatography results depletion ol the C TB pi ep on the pMB I 75()- ] 970 column remov ed all detectable reactiv ity to the pMB 1970-2360 protein T he reciprocal purification of a C I B prep that was depleted on the pMB 1970-2360 column
2 y ielded no bound antibodv w hen chiomalographcd on the pMB l 7^0- 1970 column I hese results demonstrate that all repeat reactive antibodies in the CTB polycional pool recognize a consei v ed sti uctuie that is present in non-overlapping lepeats Although it is possible that this conserv ed sti ucture represents rare conserved linear epitopes it appears more hkelv that the neuti ahzmg antibodies recognize a specific protein conformation This conclusion vvas also
30 suggested bv the results of Western blot hy bridization analy sis ol CTB reactiv ity to these iecombinant proteins
W estern blots ol 7 5% SDS-PAGE gels loaded and electrophoresed w ith defined quantities of each recombinant protein were probed with the C T B polvclonal antibodv preparation. The blots were prepared and developed with alkaline phosphatase as described in Example 3 The results are shown in Figure 24
Figure 24 depicts a comparison of immunoreactivity of IgY antibody raised against either native or recombinant toxin B antigen Equal amounts of pMB1750-1970 (lane 1), pMB 1970-2360 (lane 2). pPB 1850-2360 (lane 3) as well as a serial dilution of pPB 1750-2360
(lanes 4-6 comprising IX.1/lOX and 1/lOOX amounts, respectively) proteins were loaded in duplicate and resolved on a 75% SDS-PAGE gel The gel vvas blotted and each half was hybridized with PEG prep IgY antibodies from chickens immunized with either native CTB or pPB1750-2360 protein Note that the full-length pMB1750-1970 protein was identified only by antibodies reactive to the recombinant protein (arrows)
Although the CTB prep reacts with the pPB 1750-2360. pPBl 850-2360. and pMB1970- 2360 proteins, no reactivity to the pMB1750-1970 protein vvas observed (Figuie 24) Given that all repeat reactive antibodies can be bound by this protein during alfinity chromatography. this result indicates that the piotein cannot fold properly on V, estcrn blots Since this eliminates all antibody reactivity, it is unlikely that the repeat reactive antibodies the CTB piep recognize linear epitopes ITus may indicate that in order to induce protective antibodies, iecombinant toxin B protein will need to be properly lolded
c) Generation And Evaluation Of Antibodies Reactive To Recombinant Toxin B Polypeptides i) Generation Of Antibodies Reactive l
Recombinant Toxin B Proteins
Antibodies against recombinant proteins were generated in egg laving Eeghom hens as described in Example 13 Antibodies were raised | using 1 teunds ad|uvant (Gibco) unless otherwise indicated] against the following recombinant proteins 1) a mixture ot Interval 1+2 proteins (see Figure 18).2) a mixture of interval 4 and 5 proteins (see Figure 18).3) PMB1970-2360 protein: 4) pPB1750-2360 protein.5) pMB! 750-2360.6) pMBl 750-2360 [Iiteπnax adiuvant (Vaxcell)].7) pMBl 750-2360 (Gerbu ad ivant (Biotech)] 8) pMBpl750- 2360 piotein: 9) pPBl 850-2360. and 10) pMB1850-2360 Chickens were boosted at least 3 times with recombinant protein until FI ISA leactivitv [using the protocol described in b) above with the exception that the plates were coated with pPB1750-2360 protein] of polycional PEG preps was at least equal to that oi the CTB polycional antibody PFG prep EEISA titers were determined for the PI G preps trom all of the above immunogens and were found to be comparable ranging from 1:12500 to 1:62500 High titers were achieved in all cases except in 6) pMBl 750-2360 in which strong titers were not observed using the Titermax adjuvant, and this preparation was not tested further
ii) Evaluation Of Antibodies Reactive To
Recombinant Proteins By Western Blot Hybridization
Western blots of 75% SDS-PAGE gels, loaded and electrophoresed with defined quantities ol recombinant protein (pMB 1750- 1970, pPB I 850-2360. and pMB 1970-2360 proteins and a serial dilution of the pPB1750-2360 to allow quantification ot reactivity), were probed with the CIB. pPBl 750-2360. pMB 1750-2360 and pMB 1970-2360 polycional antibodv preparations (trom chickens immunized using Ficunds adjuvant) The blots weie piepaied and developed with alkaline phosphatase as described above in b)
\s shown in figure 24. the CTB and pMB1970-2360 preps reacted stiongly with the pPBl 7^0-2360. pPB1850-2360. and pMB 1970-2360 proteins while the pPB1750-2360 and pMB 1970-2360 (Gerbu) preparations reacted strongly with all four proteins Ihe Western blot icactiviiy ol the pPBI750-2360 and pMB1970-2360 (Gerbu) preparations were equivalent to that ot the CTB preparation, while reactivity of the pMB1970-2360 preparation was ' 10% that ot the C I prep Despite equivalent ELISA reactivities only weak reactivity (apptoximately 1%) to the recombinant proteins were observed in PEG preps from two independent groups immunized with the pMBl 750-2360 protein and one group immunized with the pMBl 750-2360 preparation using Freunds adμivant purification was utilized to determine it this difference in immunoreactivity by Western blot analysis reflects differing antibody titers Fifty ml 2X PEC3 preparations trom chickens immunized with either pMB 1750-2360 or pMB 1970-2360 protein were chromatographed on the pPBl 750-2360 affinity column from b) above, as described The yield ol affinity purified antibody (% total protein in preparation) was equivalent to the yield obtained liom a CTB PEG preparation in b) above Thus, differences m W-estern reactivity leflect a qualitative difference in the antibody pools, rather than quantitative diiteiences.. rhese icsults demonstrate that ceitain recombinant proteins are moie effective at generating high affinity antibodies (as assaved bv Western blot hybridization) iii) In Vivo Neutralization Of Toxin B Using
Antibodies Reactive To Recombinant Protein
Ihe in vivo hamster model [described in Examples 9 and 14(b)] was utilized to assess the neutralizing ability oi antibodies raised against recombinant toxm B pioteins The results fiom three expeπments are shown below in T bles 27-29
The ability of each immunogen to neutralize toxin B in
Figure imgf000130_0001
has been compiled and is shown in 1 able 30 As predicted from the recombinant prote -CTB premix studies (Table 24) onlv antibodies to Interval 3 (1750-2366) and not the other regions of toxin B (/ e intervals 1-5) are protective Unexpectedly, antibodies generated to 1NT-3 region expressed in pMAI vector (pMB 1750-2360 and pMpB 1750-2360) using Freunds adiuvant were non- neutralizing I his observation is reproducible since no neutiahzation vvas observed in two independent immunizations with pMB 1750-2360 and one immunization with pMpB1750- 2360 Ihe tact that 5X quantities ol affi itv purified toxin B repeat specific antibodies tiom pMBl 7s()-2360 PI G preps cannot neutralize toxin B while IX quantities ot atfinitv purified anti-C I B antibodies can (Table 28) demonstrates that the differential ability of CIB antibodies to neutralize toxin B is due to qualitative rather than quantitative dilferences in these antibodv preparations Only when this region was expressed in an alternative vector (pPBl 7^0-2360) ot using an alternative adiuvant with the pMBl 7^0-2360 piotem weie neutralizing antibodies generated Importantly antibodies laised using 1 reunds adiuvant to pPB1850-2360 which contains a tiagment that is onlv 100 am o acids smallei than iecombinant pPB 1750-2360 are unable to neutralize toxin B in vivo ( fable 27) note also that the same veetoi is used loi both pPB 1850-2360 and pPB I 7^0-2360
TABLE 27
In Vivo Neutralization Of Toxin B
Figure imgf000131_0002
( difficile toxin B (CTB) (at 5 μg/ml. 25 μg total. Tech Lab) at letli.il concentration to hamsteis is added to antibody and incubated for one hour at 37°C Alter incubation tins mixture is miected intraperiioneailv (IP) into hamsters f ach treatment group received toxin premixed with antibody raised against the indicated protein, as a 4λ antibodv PLG piep
Ihe numbers m eacli group represent numbers of hamsteis dead oi alive 2 hours post IP admmistiation ot
Figure imgf000131_0001
mixture
TABLE 28
In I no Neutralization OfToxin B Usinsi Affinitv Purified Antibodies
Figure imgf000131_0003
C difficile toxin B (CTB) (at 5 μg'inl. 25 μg total. Tech Lab) at lethal concentration to hamsteis is added to antibodv and incubated for one hour at 37°C After incubation. I ml ol this mixture is miected intrapeπtoneally (IP) into hamsters Fach treatment group received toxin premixed with antibody raised against the indicated protein, as either (I ) 4X antibody PEG prep or (2) affinity purified antibody (on a pPB 1750-2360 resin), either I 5 mg/group (anti-pMB 1750-2360 and anti-p B 1970-2360. used undiluted affinitv purified antibodv) or 350 μg gioup (anti-CTB repeat specific, used 1/5 diluted anti-CIB antibodv)
The numbers in eacli group represent numbers of hamsters dead or alive 2 hr post-IP admmistiation of to.xin/antibodv mixture TABLE 29
Generation Of Neutralizing Antibodies Utilizing The Gerbu Adiuvant
Figure imgf000132_0001
( ' difficile loxin B (CTB) (Tech Lab) at lethal concentralion to hamsters is added to antibodv and incubated lor one hour at 37°C. After incubation this mixture is miected intraperiioneailv (IP) into hamsters, t ach treatment group received toxin premixed with antibody raised against ihe indicated protein, as a 4X antibody PEG prep
I lie numbers in each group represent numbers ol hamsters dead oi alive.2hιs post II' administration ot toxin antibodv mixture
TABLE 30 in Vivo Neutralization OfToxin B
Figure imgf000133_0001
I itlier PIG pieparation (PFG) ot affinity purified antibodies (AP)
N es denotes complete neutralization (05 dead) while no" denotes no neutiahzation (55 dead) ol toxin B 2 houis post-administration of mixture
\Λ denotes not applicable
Ihe pPB 1750-2360 antibody pool confers significant in vivo protection, equivalent to that obtained with the affinity purified CTB antibodies. This correlates with the observed high affinitv of this antibody pool (relative to the pMBl 750-2360 or pMB1970-2360 pools) as assayed bv Western blot analysis (Figure 24). These results provide the first demonstration that in vivo neutralizing antibodies can be induced using recombinant toxin 13 protein as immunogen
The failure of high concentrations of antibodies raised against the pMBl 750-2360 protein (using Freunds adiuvant) to neutralize, while the use of Geibu adiuvant and pMB 1750-2360 protein generates a neutralizing response, demonstrates that conformation or presentation of this protein is essential for the induction of neutralizing antibodies. These results are consistent with the observation that the neutralizing antibodies produced when native CTB is used as an immunogen appear to recognize conformational epitopes [see section b) above]. This is the first demonstration that the conformation or presentation of recombinant toxin B protein is essential to generate high titers of neutralizing antibodies.
EXAMPLE 20
Determination Of Quantitative And Qualitative
Differences Between pMB l 750-2360. pMB l 750-2360 (Gerbu)
Or pPB 1750-2360 IgY Polycional Antibodv Preparations
I n Fxample 1 . it vvas demonstrated that toxin B neutralizing antibodies could be generated using specific recombinant toxin B proteins ( pPB l 750-2360) or specific adjuvants. Antibodies raised against pMB l 750-2360 were capable of neutralizing the enterotoxin effect of toxin B w hen the recombinant protein was used to immunize hens in conjunction with the Gerbu adjuvant, but not when Freunds adjuvant was used. To determine the basis for these antigen and adjuvant restrictions, toxin B-specific antibodies present in the neutralizing and non-neutralizing PI'G preparations were isolated by affinity chromatography and tested for qualitativ e or quantitative differences. The example involv ed a ) purification of anti-toxin B specific antibodies from pMB l 750-2360 and pPB ! 750-2360 PFG preparations and b) in vivo neutralization of toxin B using the affinity purified antibody.
a ) Purification Of specific Antibodies From pMB 1750-2360 And pPBl 750-2360 PEG Preparations
To puri fy and determine the concentration of specific antibodies (expressed as the percent oϊ total antibody) within the pPB l 750-2360 (Freunds and Gerbu) and pPB l 750-2360
PHG preparations, defined quantities of these antibody preparations were chromatographed on an affinity- column containing the entire toxin B repeat region ( pPB l 750-2360). The amount of affinity- purified antibody vvas then quantified.
An affinity' column containing the recombinant toxin B repeat protein. pPB l 750-2360. was made as follow s. Four ml of PBS-washed Actigel resin (Sterogene) was coupled with 5 mg of pPB l 750-2360 affinity purified protein (dialyzed into PBS: estimated to be greater than 95% full length lusion protein) in a 1 5 ml tube ( Falcon) containing 1 /10 final volume Aid- coupling solution ( 1 M sodium cyanoborohydride). Aliquots of the supernatant from the coupling reactions, beloie and aitet coupling, were assessed bv Coomassie staining of 75% SDS-PAGC gels Based on protein band intensities, greater than 95% (approximately 5 mg) of recombinant protein was coupled to the resin The coupled resin was poured into a 10 ml column (BioRad) washed extensivelv with PBS pie-elutcd with 4M guanidine-HC 1 (in 10 " m liis-HCl pH 80 0005% thimerosal) and re-equilibrated in PBS and stored al 4°C
Ahquots ot pMB 1750-2360 pMBl 750-2360 (Gerbu) oi pPB 1750-2360 IgY polycional antibodv preparations (PEG preps) were affinity purified on the above column as follows The column was attached to an I 'V monitor (ISCO) and washed with PBS r ortv ml ahquots ol 2X PI G pieps (filter sterilized using a 045 μ filter and quantified bv ()[),, before
10 chromatogiaphv ) vvas applied Ihe column was washed with PBS until the baseline was ic- estabhshed (the column flow-through vvas saved) washed with BBSTween to elute nonspecificallv binding antibodies and le-equihbrated with PBS Bound antibodv vvas eluted liom the column in 4M guanidine-HCI (in 10 mM Iiis-HCL pH 80 0005% thimerosal) and the entne elution peak collected in a 15 ml tube (falcon) The column vvas re-equilibrated
I ^ and the column eluate le-chromatographed as described above The antibodv pieparations weie quantified bv I IV absorbance (the elution butter was used to zeio the spectiophotometci ) Approximatelv 10 fold higher concentrations of total purified antibody was obtained upon elution ol the lust chiomatographv pass lelative lo the second pass Ihe low v leid tiom the second chromatography pass indicated that most of the specific antibodies 0 were removed bv the fust round ol chromatography
Pools ot affinity punfied specific antibodies were prepared bv dialysis ol the column cTutes altei the fust column chromatogiaphv pass for the pMBl 7^0-2360 pMBl 750-2360 (Geibu) oi pPBl 750-2360 IgY polvclonal antibodv preparations The eiutes were collected on ice and immediately dialvzed against a 100-fold volume ot PBS at 4°C loi 2 his fhe
25 samples weie then dialvzed against 1 changes of a 65-told volume of PBS at 4°C Dialvsis vvas pertoimed toi a minimum ol 8 hrs per change of PBS The dialvzed samples were collected centrifuged to remove insoluble debris, quantified bv OD w and stored at 4°C
The percentage ot toxin B repeat-specific antibodies present in each pieparation was determined using the quantifications ot antibodv yields trom the first column pass (amount ot
30 specific anlibodv lecovered after fiist pass/total protein loaded) Ihe yield oi lepeat-specific atfinitv purified antibodv (expressed as the percent of total piote in ihe piepaiation) in 1) the pMBl 750-2360 PLG prep was approximately 05%.2) the pMBl 7^0-2360 (Gerbu) piep was approximately 23% and 3) the pPBl 750-2360 prep was appioximatelv 04%
- ι: Purification of a CTB IgY polycional antibody preparation on the same column demonstrated that the concentration of toxin B repeat specific antibodies in the CTB preparation was 0.35%.
These results demonstrate that 1 ) the use of Gerbu adjuvant enhanced the titer of specific antibody produced against the pMBl 750-2360 protein 5-fold relative to immunization using Freunds adjuvant, and 2) the differences seen in the in vivo neutralization ability of the pMB I 750-2360 (not neutralizing) and pPB 1750-2360 (neutralizing ) and CTB (neutralizing) P G preps seen in Example 19 vvas not due to differences in the titers of repeat-specific antibodies in the three preparations because the titer of repeat-specific antibodv vvas similar for all three preps; therefore the differing ability of the three antibody preparations to neutralize toxin B must reflect qualitative differences in the induced toxin B repeat-specific antibodies. To confirm that qualitative differences exist between antibodies raised in hens immunized with different recombinant proteins and/or different adjuvants, the same amount of affinity- puri fied anti-toxin B repeat (aa 1 870-2360 of toxin B ) antibodies from the different preparations vvas administered to hamsters using the in vivo hamster model as described below .
b) /// vivo Neutralization Of Toxin B Using Affinity Purified
Antibody
The in vivo hamster model was utilized to assess the neutralizing ability of the affinity purified antibodies raised against ecombinant toxin B proteins purified in (a ) abov e. As well, a 4X IgY PEG preparation from a second independent immunization utilizing the pPB 1 750- 2360 antigen with Freunds adjuv ant vvas tested for in vivo neutralization. The results are shown in fable 3 1 .
The results shown in T able 3 1 demonstrate that: 1 ) as shown in Example 19 and reproduced here. 1 .5 mg of affinitv purified antibody from pMBl 750-2360 immunized hens using Freunds adjuvant does not neutralize toxin B in vivo. However. 300 μg of affinitv purified antibodv from similarly immunized hens utilizing Gerbu adjuvant demonstrated complete neutralization of toxin B in vivo. This demonstrates that Gerbu adjuvant, in addition to enhancing the titer of antibodies reactive to the pMB l 750-2360 antigen relative lo Freunds adjuvant (demonstrated in ( a) above), also enhances the vield of neutralizinu antibodies to this antigen, greater than 5 fold. 2) Complete in vivo neutralization of toxin B was observed with 15 mg of affinity purified antibodv from hens immunized with pPBl 750-2360 antigen, but not with pMB 1750-2360 antigen, when Freunds adjuvant was used This demonstrates, using standardized toxin B repeat-specific antibody concentrations, that neutralizing antibodies were induced when pPBl 750-2360 but not pMB 1750-2360 was used as the antigen with I reunds adjuvant
3) Complete in vivo neutralization was obseived with 300 μg ot pMBl 750-2360 (Geibu) antibodv. but not with 300 μg oi pPB1750-2360 (I reunds) antibodv
I hus the pMB175O-2360 (Gerbu) antibody has a lughei liter ot neutralizing antibodies than the pPBl 750-2360 (I reunds) antibodv
4) C omplete neutralization ol toxin B was observed using 300 μg ol CIB antibodv [affinity purified (AP)] but not 100 μg CTB antibodv (AP oi PFG piep) This demonstiates that gieater than 100 μg ol toxin B lepeat-speciflc antibodv (anti-CIB) is necessary to neutralize 25 μg toxin 13 in vivo in this assay, and that affinity purified antibodies specific to the toxin B repeat interval neutiahze toxm B as effectively as the PFP prep ol IgY laised against the enure CTB protein (shown in this assay)
5) \s was observed with the initial pPB1750-2360 (IgY) PLG prepaiation (I xample 19) complete neutralization was observed with a IgY' PFG piepaiation isolated from a second independent group of pPBl 750-2360
(1 reunds) immunized hens Fins demonstrates that neutralizing antibodies are icpioduciblv pioduced when hens are immunized with pPB175()-2360 protein utihziim 1 reunds adiuvant
TABLE 31
In vivo Neutralization Ot Toxin B Usm Affinitv Piuitled Antibodies
Figure imgf000138_0001
( difficile loxin B (C FB) ( lech Lab) at lethal concentration to hamsters (25 μu) was added lo (h antibodv (amount ol specific antibodv is indicated) and incubated lot one hour at ^ nC Λltei incubation this mixture was miected IP into hamsters (I *> total mix miected pci hamstei I Fach treatment gioup received toxin premixed vwth antibodv taiscd auainst (he indicated piotein (G »eιbιι adμivant I "Freunds adiuvant) indicates Ihe antibodv was l 4\ l_> PI G piep indicates the antibodv was affinitv punfied on i pPBI85()-23(>() lesin and indicates that the antibodv vvas a IX IgY PIG prep
The numbers in each gioup repiesent numbets ol hamsteis dead or alive 2 hrs post IP admmistiation ot toxin antibodv mixtuie
EXAMPLE 21
Diagnostic I nzvme Immunoassay s lor C difficile loxins A Λnd B
Ihe ability ol the iecombinant toxin proteins and antibodies laised against these iecombinant proteins (described in the above examples) to loi in the basis ol diagnostic assays loi the detection ot clostridial toxin in a sample vvas examined Two immunoassay toi mats were tested to quantitatively detect ( difficile toxm and toxin B trom a biological specimen Ihe fust toi mat involved a competitive assav in which a fixed amount ot iecombinant toxin A oi B was immobilized on a solid suppott (e g , miciolilei plate wells) toilovved bv the addition ol a toxin-containing biological specimen mixed with affinity - punfied oi PIG hactionated antibodies against recombinant toxin A oi B II toxm is piesent in a specimen, this toxm will compete with the immobilized iecombinant tox protein tor binding to the anti-reeombinant antibody thereby reducing the signal obtained following the addition of a reporter reagent The reporter reagent detects the presence of antibody bound to the immobilized toxin protem
In the second tormat. a sandwich immunoassay was developed using affinity-purified antibodies to recombinant toxin Λ and B Ihe affinity -purified antibodies to recombinant toxm Λ and B were used to coat microtiter wells instead of the iecombinant polypeptides (as was done in the competitive assav format) Biological samples containing toxin A oi B were then added to the wells followed bv the addition ol a repoiler reagent to detect the presence ol bound toxin in the well
a) Compctitiλe Immunoassay For The Detection Of C. difficile
Toxin
Recombinant tox A oi B vvas attached to a solid support bv coating % well micioiitei plates with the toxin protem at a concentration ol 1 μg/ml in PBS The plates were incubated overnight at 2-8°C The following morning, the coating solutions were removed and ihe remaining piotein binding sites on the wells were blocked bv filling each well with a PBS solution containing 05% BSA and 005% Iween-20 Native ( difficile toxin A oi B ( lech I ab) vvas diluted to 4 μg/ml in stool extracts from healthy Syrian hamsters (Sasco) Ihe slool extracts were made bv placing fecal peliets in a 15 ml centrifuge tube. PBS was added at 2 ml/pellet and the tube vvas vortexed to create a uniform suspension The tube vvas then cenlπfuged at 2000 rpm for 5 mm at room temperatuie Ihe supernatant vvas removed, this compiises the stool extract 1 ιfty μl of the hamster stool extract vvas pipetted into each well ol the microtiter plates to serve as the diluent tor senal dilutions ot the 4 μg/ml tox samples One hundied μl ot the toxin samples at 4 μg/mi vvas pipetted into the first row ol wells in the microtiter plate, and 50 μl aliquots were removed and diluted serially down the plate in duplicate An equal volume ot affinity purified anti-reeombinant toxin antibodies [1 ng/well ol anti-p Al 870-2680 antibodv was used toi the detection ot toxin A.05 ng/well ot anti-pMBl 750-2360((ieιbu) was used for the detection ot toxin B] weie added to appropriate wells, and the plates were incubated at room temperature lot 2 hours with gentle agitation Wells seivmg as negative control contained antibodv but no native toxin to compete lor binding
Unbound toxin and antibody were removed bv washing the plates 3 to 5 times with PBS containing 005% "Ivveen-20 Following the wash step. 100 μl of rabbit anti-chicken Igϋ antibody conjugated to alkaline phosphatase (Sigma) w as added to each well and the plates were incubated for 2 hours at room temperature. The plates were then washed as before to remove unbound secondary antibody. Freshly prepared alkaline phosphatase substrate ( 1 mg/ml p-nitrophenyl phosphate ( Sigma) in 50 mM NaXO ,. pl l 9.5: 10 mM MgCF) was added to each well. Once sufficient color developed, the plates were read on a Dynatech
MR700 microtiter plate reader using a 410 nm filter.
The results are summarized in Tables 32 and 33. For the results show n in Table 32. the wells w ere coated with recombinant toxin A protein ( pMA I 870-2680 ). Fhe amount of native toxin A added (present as an addition to solubili ed hamster stool ) to a given well is indicated ( 0 lo 200 ng). Antibody raised against the recombinant toxin A protein. pMA 1870-
2680. w as affinity purified on the an affinity column containing p A 1 870-2680 ( described in Fxample 20). As shown in Table 32. the recombinant toxin A protein and affinity-purified antitoxin can be u.sed for the basis of a competitive immunoassay tor the detection of toxin A in biological samples. Similar results were obtained using the recombinant toxin B. pPB 1 750-2360. and antibodies raised against pMB 1750-2360((ϊerbu ). For the results show n m Table 33. the wells were coated with recombinant toxin B protein ( pPB l 750-2360). The amount of native toxin B added ( present as an addition to solubilizcd hamster stool ) to a given well is indicated (0 to 200 ng). Antibody raised against the recombinant toxin B protein. pMB 1 750- 2360(( ierbu). was affinity purified on the an affinity column containing pPB I 850-2360
( described in F ample 20 ). As shown in Fable 33. ihe recombinant toxin 1-5 protein and affinity-purified antitoxin can be used for the basis of a competitiv e immunoassay for the detection of toxin B in biological samples.
I n this competition assay, the reduction is considered significant ov er the background levels at all points: therefore the assay can be used to detect samples containing less than 12.5 nu toxin A/well and as little as 50- 100 nu toxin B/well.
TABLE 32
Competitive Inhibition Of Anti-C difficile Toxin A Bv Native Toxin A
Figure imgf000141_0001
TABLE 33
( ompetitive Inhibition Of Λnti-C difficile Toxin B Bv N.uivc loxin B
Figure imgf000141_0002
1 "hese competitive inhibition assays demonstrate that native C difficile toxins and iecombinant ( difficile toxin proteins can compete for binding to antibodies raised against iecombinant (' difficile toxins demonstrating that these anti-recombinant toxin antibodies ptovi e effective diagnostic reagents
b) Sandwich Immunoassay For The Detection Of C. difficile
Toxin
Affinity -purified antibodies against iecombinant toxin A oi toxin B were immobilized to 96 well microtiter plates as follo s The wells were passively coated overnight at 4°C with affinity purified antibodies rai.scd against either pMA 1870-2680 (toxin A) or pMB1750- 2360(Gerbu) (toxin B). The antibodies were affinity purified as described in Example 20. The antibodies were used at a concentration of 1 μg/ml and 100 μl was added to each microtiter well. I he wells were then blocked with 200 μl of 0 5% BSA in PBS for 2 hours at room temperature and the blocking solution was then decanted. Stool samples from healthy Syrian hamsters were resuspended in PBS, pi I 7.4 (2 ml PBS/stool pellet vvas used to resuspend the pellets and the sample was centrifuged as described above). The stool suspension vvas then spiked with native ( '. difficile toxin A or B ('Fech Lab) al 4 μg/ml. The stooi suspensions containing toxin (either toxin A or toxin B) were then serially diluted twofold in stool suspension without toxin and 50 μl was added in duplicate to the coated microtiter wells. Wells containing stool suspension without toxin served as the negative control.
Fhe plates were incubated for 2 hours at room temperature and then were w ashed three times w ith PBS. One hundred μl of either goat anti-native toxin A or goat anti-native toxin B ( l ech Fab) diluted 1 : 1000 in PBS containing 1 % BSA and 0.05% I ween 20 was added to each well . The plates were incubated for another 2 hours at room temperature.
Fhe plates w ere then w ashed as before and 100 μl of alkaline phosphatase-conjugated rabbit anti-goat IgC i (Cappel. Durham. N.C.) vvas added at a dilution of 1 : 1000. T he plates were incubated for another 2 hours at room temperature, The plates were washed as before then developed by the addition of 100 μl/well of a substrate solution containing I mg'inl p- nitrophenyl phosphate ( Sigma) in 50 mM Na C -„ pFI 9.5: 10 mM MgCF. Fhe absorbance of each w ell was measured using a plate reader ( Dynatech ) at 410 nm. The assay results are show n in Fables 34 and 35.
TABLE 34
C diffic ile l ox in Λ Detect ion I n Stool Using Affinitv -Purified Ant ibodies Against Tox in A
Figure imgf000142_0001
TABLE 35
( ' difficile Toxin B Detection In Stool Using Affimty-Put ified Antibodies Against Toxin B
Figure imgf000143_0001
The results shown in Fables 34 and 35 show that antibodies raised against recombinant loxin A and tox B fragments can be used to detect the presence ol ( ' diffic ile toxin in stool samples I hese antibodies form the basis for a sensitiv e sandw ich immunoassay which is capable of detecting as little as 6.25 ng of either toxin A or B in a 50 μl stool sample As show n above in I bles 34 and 35. the background for this sandw ich immunoassay is exti emely low . therefore, the sensitiv ity of this assay is much lower than 6 25 ng toxin/well. I t is likely that toxin lev els of 0 5 to 1 .0 pg/well could be detected by this assay
The results shown above in Tables 32-35 demonstrate clear utility ot the recombinant i cagents in C difficile toxin detection systems.
EXAMPLE 22
Construction And Expression Of C hoiulinum C Fragment 1 usion Proteins
Fhe ( ' botulinum ty pe A neurotoxin gene has been cloned and sequenced [Thompson. ei al . Eur .1. Biochem. 1 89:73 ( 1990)] The nucleotide sequence of the toxin gene is av ailable from the EMBL/GenBank sequence data banks under the accession number X52066: the nucleotide sequence of the coding region is listed in SEQ ID NO 27. I he amino acid sequence of the ( ' hoiulinum ty pe A neurotoxin is listed in SEQ I D NO.28. T he ty pe A neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds. I he 50 kD carboxy- termmal portion of the heavy chain is referred to as the C fragment or the I I, domain.
Previous attempts by others to express polypeptides compπsing the C fragment oϊ C botulinum type A toxm as a nativ e polypeptide (e g., not as a fusion protein) in £ coh have been unsuccessful [H.F. LaPenotiere. et al. in Botulinum and Tetanus Neuroioxins. DasGupta. Ed.. Plenum Press. New York ( 1993). pp. 463-466]. Expression of the C fragment as a fusion with the E. coli MBP was reported to result in the production of insoluble protein (H.F. LaPenotiere. et al.. supra). In order to produce soluble recombinant C fragment proteins in E. coli. lusion proteins comprising a synthetic C fragment gene derived from the C hoiulinum type A toxin and either a portion of the ( '. difficile toxin protein or the MBP were constructed. This example involved a) the construction of plasmids encoding C fragment fusion proteins and b) expression of C. botulinum C fragment fusion proteins in E. coh.
a) Construction Of Plasmids Encoding C Fragment Fusion
Proteins In Example 1 1. it was demonstrated that the C difficile toxin A repeat domain can be efficiently expressed and purified in E. coli as either native (expressed in the pE'F 23a vector in clone pPA 1 870-2680) or lusion (expressed in the pMALc vector as a fusion with the E. coli M BP in clone pMA I 870-2680) proteins. Fusion proteins comprising a fusion between the MBP. portions of the ( ' difficile toxin A repeat domain ( show n to be expressed as a soluble fusion protein ) and the C fragment of the C botulinum type A loxin were constructed. A fusion protein comprising the C fragment of the C hoiulinum type A toxin and the MBP was also constructed.
Figure 25 provides a schematic representation of the botulinal fusion proteins along w ith the donor constructs containing the C. difficile toxin A sequences or ( ' botulinum C Iragment sequences which were used lo generate the botulinal fusion proteins. In Figure 25. ihe solid boxes represent C difficile toxin A gene sequences, the open boxes represent ( ' hoiulinum C fragment sequences and the solid black ovals represent the E. coli MBP. When the name for a restriction enzyme appears inside parenthesis, this indicates that the restriction site was destroyed during construction. An asterisk appearing with the name for a restriction enzyme indicates that this restriction site vvas recreated at the cloning junction.
I n Figure 25. a restriction map of the pMA I 870-2680 and pPA l 100-2680 constructs ( described in E.xample 1 1 ) which contain sequences derived from the ( '. difficile toxin A repeat domain are shown: these constructs were used as the source of C difficile toxin A gene sequences f r the construction of plasmids encoding fusions between the ( '. hoiulinum C fragment gene and the ( ' difficile toxin A gene. Fhe pMA 1870-2680 expression construct expresses high levels of soluble, intact fusion protein (20 mg/liter culture) which can be affinity purified on an amylose column ( purification described in Example 1 I d).
The pAlterBot construct (Figure 25) was used as the source of C hoiulinum C fragment gene sequences for the botulinal fusion proteins. pAlterBot was obtained from J. Middlebrook and R. Lemley at the U.S. Department of Defense. pAlterBot contains a synthetic ( ' hoiulinum C fragment inserted in to the pALTER- l i1 vector ( Promega). This synthetic C fragment gene encodes the same amino acids as does the naturally occurring C I ragment gene. The naturally occurring C fragment sequences, like most clostridial genes, are extremely AT rich (Thompson et al.. supra). This high A/T content creates expression difficulties in E. coh and yeast due to altered codon usage frequency and fortuitous polyadenylation sites, respectively. In order to improve the expression of C fragment proteins in /-. coli. a synthetic version of the gene was created in w hich the non-preferred codon were replaced with prelerred codons.
The nucleotide sequence of the ( ' hoiulinum C fragment gene sequences contained w ithin pAlterBot is listed in SEQ I D NO:22. The first six nucleotides ( ATGGCT) encode a methionine and alanine residue, respectively. These two amino acids result from the insertion of the ( ' hoiulinum C fragment sequences into the pALTER® vector and provide the initiator methionine residue. The amino acid sequence of the ( '. hoiulinum C fragment encoded by the sequences contained within pAlterBot is listed in SEQ I D NO:23. The first two amino acids ( Met Ala ) are encoded by vector-derived sequences. From the third amino acid residue onward ( Arg ). the amino acid sequence is identical to that found in the C botulinum type A toxm gene.
I he pMA I 870-2680. pPA l 1 0-2680 and pAlterBot constructs were used as progenitor plasmids to make expression constructs in which fragments of the ( ' difficile toxin A repeat domain were expressed as genetic fusions with the C. hoiulinum C fragment gene using the pMΛF-c expression vector ( New England BioEabs). The pMAL-c expression vector generates fusion proteins which contain the MBP at the amino-terminal end of the protein. A construct. pMBot. in which the C. hoiulinum C fragment gene was expressed as a fusion with only the MBP was constructed ( Figure 25). Fusion protein expression vvas induced from E. coli strains harboring the above plasmids. and induced protein was affinity purified on an amvlose resin column. i) Construction Of pBIueBot
In order to facilitate the cloning of the ( ' hoiulinum C fragment gene sequences into a number of desired constructs, the botulinal gene sequences were removed from pAlterBot and were inserted into the pBluescπpt plasmid (Stratagene) to generate pBIueBot (F igure 25). pBIueBot was constructed as follows. Bacteria containing the pAlterBot plasmid were grown in medium containing tetracycline and plasmid DNA was isolated using the QlAprep-spm Plasmid Kit ( Qiagen ). One microgram of pAlterBot DNA w as digested w ith ,\ col and the resulting 3 " recessed sticky end was made blunt using the Klenow fragment of DNA polymerase I ( here after the Klenow fragment) I e pAlterBot DNA vvas then digested with Hindl ll to release the botulinal gene sequences ( the Bot insert) as a blunt ( filled Xcol site)- l/tndl l l fragment. pBluescript vector DNA was prepared by digesting 200 ng of pBluescπpt DNA w ith Smal and Hindl ll. fhe digestion products from both plasmids w ere resolved on an agarose gel. Fhe appropriate fragments were remov ed I rom the gel. mixed and purified utilizing the Prep-a-Gene kit ( BioRad ) The eluted DNA vv as then ligated using 14 DNA ligase and used to transform competent DH5 cells ( Gibco-BRI ) Host cells were made competent lor transformation using the calcium chloride protocol ol Sambrook et al.. supra at 1 .82- 1 S i Recombinant clones were isolated and confirmed by restriction digestion using standard recombinant molecular biology techniques ( Sambrook et al. supra) I he resultant clone. pBIueBot. contains several useful unique restriction sites flanking the Bot insert ( / e . the ( ' hoiulinum C fragment sequences derived from pAlterBot) as show n in 1 igure 25
ii) Construction Of C. difficile / C. botulinum I
MBP Fusion Proteins
Constructs encoding fusions between the C difficile toxin A gene and the C botulinum C Iragment gene and the MBP were made utilizing the same ecombinant DNA methodology outlined above: these fusion proteins contained varying amounts of the C difficile toxm A repeat domain.
Fhe pMABot clone contains a 2.4 kb insert deriv ed Irom the ( ' diffic ile tox A gene f used to the Bot insert ( i e. the C botulinum C fragment sequences deriv ed Irom pAlterBot) pMABot ( Figure 25) vvas constructed by mixing gel-purified DNA Irom \otll Hindlll digested pBIueBot ( the 1 .2 kb Bot fragment). SpellNotl digested pPA l 100-2680 (the 2.4 kb ( ' difficile toxm A repeat fragment) and Xhall Hindl l l digested pMAI -c v ector Recombinant clones were isolated, confirmed by restriction digestion and purified using the QIAprcp-spin Plasmid Kit (Qiagen) This clone expresses the toxin A repeats and the botulinal C fragment protein sequences as an in-frame fusion with the MBP
The pMCABot construct contains a 1 .0 kb insert derived from the ( ' difficile toxin A gene fused to the Bot insert (/ e. the ( ' hoiulinum C fragment sequences derived from pAlterBot) pMCABot was constructed by digesting the pMABot clone with EcoRl to iemov e the 5" end of the ( ' difficile toxin A repeat (see Figure 25. the pMAI -c vector contains a EcoR] site 5' to the C difficile insert in the pMABot clone). Fhe restriction sites w ere filled and re gated together alter gel purification The resultant clone (pMCABot. 1 igui e 25 ) generated an in-trame tusion between the MBP and the remaining 3 ' portion of the ( ' difficile tox A repeat domain fused to the Bot gene
I he pMNABot clone contains the 1 kb Spell Ec oRl (filled) fragment from the C diffic ile toxin A repeat domain (derived from clone pPA l 100-2680) and the 1.2 kb ( " hoiulinum C l i agmenl gene as a Xc ol ( filled)/////7c/III fragment (deπved from pAlterBot) I hese two liagments were inserted into the pMAL-c vector digested with XhallLhndlll The two insert liagments were generated by digestion of the appropriate plasmid with EcoRl ( pPA l 100-2680) or \ c ol ( pAlterBot) lollowed by treatment w ith the Klenow f i agment After treatment w ith the Klenow I ragment. the plasmids were digested w ith the second en/yme (either Spel oi Hindll l ) All three fragments were gel purified, mixed and Prep-a-Genc purified prior to ligation f ollowing ligation and transformation, putative recombinants were analy zed by restriction analy sis, the EcoRl site was found to be regenerated at the f usion lunction. as w as predicted for a fusion between the filled EcoRl and ι\c ol sites
\ construct encoding a tusion protein between the botuhnal ( f ragment gene and the MBP gene vvas constructed ( i e . this tusion lacks any C diffic ile toxin A gene sequences) and termed pMBot I he pMBot construct was made by removal of the ( ' difficile toxin A sequences from the pMABot construct and fusing the C Iragment gene sequences to the MBP This was accomplished by digestion of pMABot DNA with Λ7//I (located in the pMALc poly lmkei 5" to the Xbal site) and Xbal (located 3' to the hotl site at the toxA-Bot f usion junction ), filling in the Xbal site using the Klenow fragment, gel purifying the desired lestrietion f i agment. and ligating the blunt ends to circularize the plasmid Following ligation and transf ormation, putativ e recombinants were analyzed by restriction mapping ot the Bot insert ( / e. the hoiulinum C fragment sequences) b) Expression Of C. botulinum C Fragment Fusion Proteins In
Large scale (1 liter) cultures of the pMAL -c vector and each recombinant construct described above in (a) were grown, induced and soluble protein tractions weie isolated as desciibed in Example 18 Ihe soluble protein extracts weie chromatographed on amylosc affinitv columns to isolate recombinant tusion protein Ihe purified recombinant lusion pioteins were analyzed bv lunning samples on SDS-PA.GE gels lollowed by Coomassie staining and bv Western blot analvsis as described [Williams el al (1994) supia] In brief, extiacts were prepared and chromatographed in column butter ( 10 mM NaP04 05 M NaCl 10 mM (3-mercaptoethanol pH 72) over an amylose resin (New England Biolabs) column, and eluted with column buffer containing 10 mM maltose as desciibed [Williams et ul ( 1994) supia] An SDS-PAGF gel containing the purified protem samples stained with Coomassie blue is shown in Figuie 26
In I igure 26 the tollovving samples were loaded I anes I 6 contain protein purified liom / coh containing the pMAL -c pPΛ1870-2680. pMABot pMNABot pMC \Bot and pMBot plasmids respectively 1 ane 7 contains broad range moleculai weight protein markers (BioRad)
Ihe piotem samples were piepaied lor electrophoresis bv mixing ^ μl ol eluted piotein with 5 μl of 2X SDS-PAGE sample buffer (0125 mM Ins-HC I pll 68 2 mM EDI A 6% SDS 20% glvceiol 0025% bromophenol blue [i-mercaptoethanol is added to 5% betore use) Ihe samples were heated to 95°C for 3 mm. then cooled and loaded on a 75% agarose SDS-PΛG1 gel Bioad lange molecular weight ptotem markers weie also loaded to allow estimation ot the MW ol identified fusion proteins After electrophoresis piotein vvas detected generally bv staining the gel with Coomassie blue
In all cases the yields weie m excess of 20 mg lusion piotem pei litei culture (see f ble 36) and with the exception ol the pMCABot protein a high percentage (i c greater than 20-50% ot lotal eluted protein) ol the eluted lusion protem was ol a MW piedicted foi the lull length tusion protem (1 iguie 26) It was estimated (bv visual inspection) that less than 10% ol the pM ABot lusion protein was expiessed as the lull length lusion piotein TABLE 36
Yield Of' Affinitv Purified C. hoiulinum C Franment / MBP Fusion Proteins
Figure imgf000149_0001
T hese results demonstrate that high level expression of intact C hoiulinum C fragment^ ' difficile toxin A fusion proteins in E. coli is feasible using the pMAL-c expression system. These results are in contrast to those reported by H. F. LaPenotiere. el al. ( 1 93 ). supra. I n addition, these results show that it is not necessary to fuse the botulinal C fragment gene to the (. '. difficile toxin A gene in order to produce a soluble fusion protein using the pMΛL-c system in /'. coli.
I n order to determine whether the above-described botulinal fusion proteins were recognized by anti-C hoiulinum toxin A antibodies. Western blots were performed. Samples containing affinit -purified proteins from /:'. coli containing the pMABot. pMCABot. pMNABot. pMBot. pMA I 870-2680 or pMALc plasmids were analyzed. SDS-PAGE gels ( 7.5%" acryiamide ) were loaded with protein samples purified from each expression construct.
A ter electrophoresis. the gels were blotted and protein transfer was confirmed by Ponceau S staining ( as described in Example 1 2b ).
Follow ing protein transfer, the blots were blocked by incubation for 1 hr at 20°C in blocking buffer [ PBST ( PBS containing 0.1 % Tvveen 20 and 5% dry milk)]. The blots were then incubated in 10 ml of a solution containing the primary antibody: this solution compri.sed a 1 /500 dilution of an anti-C hoiulinum toxin A IgY PEG prep (described in Example 3 ) in blocking buffer. The blots were incubated for I hr at room temperature in the presence of the primary antibody. Fhe blots were washed and developed using a rabbit anti-chicken alkaline phosphatase conjugate ( Boehringer Mannheim) as the secondary antibody as follows. Fhe rabbit anti-chicken antibodv was diluted to 1 μg/ml in blocking buffer ( 10 ml final volume per blot ) and the blots were incubated at room temperature for I hour in the presence of the secondary antibody. Fhe blots were then washed successiv ely with PBST. BBS-Tween and 50 mM Na.CO.. pH 9.5. The blots were then developed in freshly-prepared alkaline phosphatase substrate buffer ( 100 μg/ml nitro blue tetrazolium. 50 μg/ml 5-bromo-chloro- indolylphosphate. 5 mM MgCE in 50 mM NaXO,. pH 9.5). Development was stopped by flooding the blots with distilled water and the blots were air dried.
This Western blot analysis detected anti-C botulinum toxin reactive proteins in the pMABot. pMCABot. pMNABot and pMBot protein samples (corresponding to the predicted full length proteins identified above by Coomassie staining in Figure 26). but not in the pMA I 100-2680 or pMALc protein samples.
These results demonstrate that the relevant fusion proteins purified on an amylose resin as described above in section a) contained immunoreactive ( ' botulinum C fragment protein as predicted.
EXAMPLE 23
Generation Of Neutralizing Antibodies By Nasal Administration Of pMBot Protein
Fhe ability of the recombinant botulinal toxin proteins produced in Example 22 to stimulate a sy stemic immune response against botulinal toxin epitopes was assessed. This example involv ed: a ) the evaluation of the induction of serum IgG titers produced by nasal or oral administration of botulinal toxin-containing C. difficile toxin A fusion proteins and b) the in vivo neutralization of ( ', botulinum type A neurotoxin by anti- recombinant ( '. hoiulinum C fragment antibodies.
a) Evaluation Of The Induction Of Serum l«(ϊ l iters Produced
By Nasal Or Oral Administration Of Botulinal Toxin- Containing C. difficile Toxin A Fusion Proteins
Six gi oups containing five 6 week old CF female rats (Charles River) per group were immunized nasally or orally w ith one of the following three combinations using protein prepared in Example 22: ( I ) 250 μg pMBot protein per rat ( nasal and oral ); 2 ) 250 μg pMABot protein per rat ( nasal and oral): 3 ) 125 μg pMBot admixed with 125 μg pMA 1 870- 2680 per rat ( nasal and oral ). A second set of 5 groups containing 3 CY female rats/group were immunized nasally or orally with one of the following combinations (4) 250 μg pMNABot protein per rat ( nasal and oral ) or 5) 250 μg pMAF-c protein per rat ( nasal and oral). The fusion proteins were prepared for immunization as follows. The proteins ( in column buffer containing 10 mM maltose) were diluted in 0. 1 M carbonate buffer. pH 9.5 and administered orally or nasally in a 200 μl volume. The rats were lightly sedated with ether prior to administration. The oral dosing was accomplished using a 20 gauge feeding needle. The nasal dosing was performed using a P-200 micro-pipettor (Gilson). Fhe rats were boosted 14 days after the primary immunization using the techniques described above and were bled 7 days later. Rats from each group were lightly etherized and bled from the tail. The blood was allowed to clot at 37°C for 1 hr and the serum was collected.
The serum from individual rats was analyzed using an ELISA to determine the anti-C. hoiulinum t pe A toxin IgG serum titer. The ELISA protocol used is a modification of that described in Example 1 3c. Briefly. 96-well microtiter plates ( Falcon. Pro-Bind Assay Plates) were coated with ( '. hoiulinum type A toxoid (prepared as described in Example 3a) by placing 100 μl volumes of ( '. hoiulinum type A toxoid at 2.5 μg/ml in PBS containing 0.005% thimerosal in each well and incubating overnight at 4°C. The next morning, the coating suspensions were decanted and all wells were washed three times using PBS.
In order to block non-specific binding sites. 100 μl of blocking solution [0.5% BSA in PBS) was then added lo each well and the plates were incubated for 1 hr at 37°C. The blocking solution was decanted and duplicate samples of 1 50 μl of diluted rat serum added to the first well of a dilution series. The initial testing serum dilution was 1 :30 in blocking solution containing 0.5% Tween 20 followed by 5-fold dilutions into this solution. This was accomplished by serially transferring 30 μl aliquots to 120 μl blocking solution containing 0.5% Tween 20. mixing, and repealing the dilution into a fresh well. After the final dilution. 30 μl was removed from the well such that all wells contained 120 μl final volume. A total of 3 such dilutions were performed (4 wells total ). The plates were incubated I hr at 37°C. Following this incubation, the serially diluted samples were decanted and the wells were w ashed six times using PBS containing 0.5% Tween 20 (PBST). To each well. 100 μl of a rabbit anti-Rat IgG alkaline phosphatase (Sigma) diluted ( 1/1000) in blocking buffer containing 0.5% Tween 20 was added and the plate was incubated for 1 hr at 37°C. I he conjugate solutions were decanted and the plates were washed as described above, substituting 50 mM NaXO,. pH 9.5 for the PBST in the final wash. The plates were developed by the addition of 100 μl of a solution containing I mg/ml para-nitro phenyl phosphate (Sigma) dissolved in 50 mM NaXO,. 10 mM MgCF. pH 9,5 to each well, and incubating the plates at room temperature in the dark for 5-45 min. The absorbency of each well vvas measured at 410 nm using a Dynatech MR 700 plate reader. The results are summarized in Tables 37 and 38 and represent mean serum reactivities of individual mice.
TΛBI.F. 37
Determination ( II Λnli-C bniiilm m I
Figure imgf000152_0001
Scrum Iglj liters I oll u irm Immunization W ith ( ' hoiiilimi ( ' rraμment-('oιιlaιnιn<! l usion I'roiems
Figure imgf000152_0002
umb s represem (h a\era_e \ allies obtained Irom l o I. LISA plales standardized uuli/iπn Ihe preimmmn. uinlrol
TABLE 38
Determination OI' Ant i-C hotuhniini l pc A I'ox in Serum lyG Tilers "'ollovvinji Immunization With C hoiulinum C r- raument-Coπtaimnii Fusion Proteins 0
Figure imgf000152_0003
Fhe above FLISA results demonstrate that reactivity against the botulinal fusion 0 proteins w as strongest when the route of administration was nasal; only weak responses were stimulated when the botulinal fusion proteins were given orally . Nasally delivered pMbot and pMBot admixed with pMA I 870-2680 invoked the greatest serum IgG response. Fhese results show that only the pMBot protein is necessary to induce this response, since the addition of the pMA 1 870-2680 protein did not enhance antibody response ( Fable 37). Placement oϊ the
_ϊ> C. difficile toxin A fragment between the MBP and the ( '. botulinum C fragment protein dramatically reduced anti-bot IgG titer (see results using pMABot. pMCABot and pMNABot proteins).
This study demonstrates that the pMBot protein induces a strong serum IgG response directed against C. hoiulinum type A toxin when nasally administered.
b) //; Vivo Neutralization Of C. botulinum Type A Neurotoxin By Anti- Recombinant C. botulinum C Fragment Antibodies
The ability of" the anti-C. hoiulinum type A toxin antibodies generated by nasal administration ol" recombinant botulinal fusion proteins in rats ( Fxample 22) to neutralize ( ' hoiulinum tv pe A toxin vvas tested in a mouse neutralization model. The mouse model is the art accepted method For detection of botulinal toxins in body fluids and for the evaluation of anti-botulinal antibodies [ F.J. Schantz and D.A. Kautter. J. Λssoc. Off. Anal. Chem. 61 :96 ( 1990 ) and I nvestigational New Drug ( BB-IND-3703) application by the Surgeon General of the Department of the Army to the Federal Food and Drug Administration ). The anti-C hoiulinum type A toxin antibodies were prepared as follows.
Rats from the group given pMBot protein by nasal administration were boosted a second time with 250 μg pMBot protein per rat and serum was collected 7 days later. Serum from one rat from this group and from a preimmune rat w as tested for anti-C hoiulinum type A toxin neutralizing activity in the mouse neutralization model described below . Fhe FD„, of a solution of purified C botulinum type A toxin complex, obtained from
Dr. Fric Johnson ( University of Wisconsin Madison), was determined using the intraperitoneal ( I P ) method of Schantz and Kautter [J. Λssoc. Off. Anal. Chem. 61 :96 ( I 978 ) | using 1 8-22 gram female ICR mice and as found to be 3500 FD<„/ml. The determination of the LDW was performed as follows. A Type A toxin standard vvas prepared by dissolving purified type A toxin complex in 25 mM sodium phosphate buffer, pll 6.8 to yield a stock toxin solution of
3. 15 x 10' LD„,/mg. The Ol 7S of the solution was determined and the concentration was adjusted to 10-20 μg/ml. The toxin solution was then diluted 1 : 100 in gel-phosphate (30 mM phosphate, pl l 6.4: 0.2% gelatin). Further dilutions of the toxin solution were made as shown below in Fable 39. Two mice were injected IP with 0.5 ml of each dilution show n and the mice were observed for symptoms of botulism for a period of 72 hours. TABLE 39
Determination Of The LD< Ol Purified C hoiulinum Ivpe A loxin Complex
Figure imgf000154_0001
From the results shown in Table 39 the toxin titer was assumed lo be between 2560
1 D,,/ml and 5120 L D,„/ml (oi about 3840 I D„ ml) This value was lounded to 3500 LD 1/ml lot the sake ot calculation
Th amount ot neutralizing antibodies piesent in the serum ot rats immuni/ed nasallv with pMBot protein vvas then determined Serum liom two rats boosted with pMBot protein as desciibed above and pieimmune serum Irom one rat vvas tested as follows Ihe toxin standaid vvas diluted 1 100 in gel-phosphate to a final concentration oi 3 ) I D ( mi One millilitei ol the diluted toxin standard was mixed with 25 μl ol seru liom each ol the three its and 02 ml ol gel-phosphate Ihe mixtures were incubated at loom tempeiatuie lot 30 mm with occasional mixing Fach ot two mice were miected with IP with 05 ml ol the mixtuies Ihe mice were observed toi signs ot botulism tor 72 hr Mice receiving seium from rats immunized with pMBot protein neutralized this challenge dose Mice leceiving preimmune rat serum died m less than 24 hr
Ihe amount ot neutralizing anti-toxin antibodies present the seium of rats immunized with pMBot piotein was then quantitated Seium antibodv titiations weie pet toi med bv mixing 01 ml of each ot the antibody dilutions (see 1 able 40) with 01 ml ol a
1 10 dilution ot stock toxin solution (35 x 1()4 I Dvι/ml) with 10 ml ol gel phosphate and lniecting 05 ml IP into 2 mice per dilution Ihe mice were then observed loi signs ol botulism lor 3 davs (72 hr) Fhe results are tabulated in Table 39
Λs shown in I able 40 pMBot serum neutralized ( hoiulinum tvpe Λ toxin complex when used at a dilution of 1320 oi less A mean neutralizing v lue ot 168 II I/ml was obtained loi the pMBot seium (an UJ is defined as 10000 mouse L D\ ) I his value tianslates to a ciiculating serum titer ot about 37 lU/mg ol serum protein 1 his neutralizing titer is compaiable to the commerciallv available bottled concentrated (( onnaught 1 aboratones I td ) horse antι-( hoiulinum antiseium A 10 ml vial ot onnaught antiserum contains about 200 mg/ml of protei each ml can neutralize 750 IU of ( '. hoiulinum type A toxin. After administration of one vial to a human, the circulating serum titer of the Coπnaught preparation would be approximately 25 l U/ml assuming an average serum volume of 3 liters). Thus, the circulating anti-C botulinum titer seen in rats nasally immunized with pMBot protein ( 168 l U/ml ) is 6.7 time higher than the necessary circulation titer of anti-C. botulinum antibodv needed to be protectiv e in humans.
TABLE 40
Ouantitation Of Neutralizing Antibodies In pMBot Sera
1
Figure imgf000155_0001
Numbers represent the number of mice surviving at 72 hours which received serum taken from rats immunized with the pMBot protein.
'' These mice survived but w ere sick after 72 hr.
Fhese results demonstrate that antibodies capable of neutralizing C. botulinum type A toxin are induced when recombinant C " hoiulinum C fragment fusion protein produced in E.
"> coli is used as an immunouen.
EXAMPLE 24
Production Of Soluble C. hoiulinum C Fragment Protein Substantially Free Of Hndotoxin Contamination
30
Fxample 23 demonstrated that neutralizing antibodies are generated b\ immunization w ith the pMBot protein expressed in E. coli. These results showed that the pMBot fusion protein is a good vaccine candidate. However, immunogens suitable for use as vaccines should be pyrogen-free in addition to having the capability of inducing neutralizing
- is: antibodies Expression clones and conditions that facilitate the production ol C hoiulinum C fiagment protein for utilihzation as a vaccine were developed
The example involved (a) determination ot pvrogen content of the pMBot protein. (b) generation ol C botulinum C fragment protein liee of the MBP. (c) expression of C botulinum C f iagment protem using various expression vectors, and (d) purification ol soluble
( hoiulinum C fragment protein substantially free ot significant endotoxin contamination
a) Determination Of The Pvrogen ontent Of The pMBot
Protein I n oi der to use a recombinant antigen as a v accine in humans or other animals, the antigen preparation must be shown to be tree ot pyrogens I he most significant pvrogen present in preparations ot recombinant proteins produced gram-negative bacteria, such as t e oli is endotoxin [ 1 C Pearson. Px rogens endotoxms I AL testing and dep\ rogentaιon, ( 1985 ) Marcel Dekker. New York, pp 23-56J l o ev aluate the utilitv of the pMBot piotein as a v accine candidate, the endotoxin content in MBP fusion proteins vvas determined
I he endotoxin content ot recombinant protein samples was assav ed utilizing the I imulus assav ( I AL kit. Associates ol Cape Cod ) according to the manufacturers instructions Samples of atfinity-puπfled pMal-c protem and pMA 1870-2680 wei e found to contain high levels ol endotoxin [ '50.000 L J/mg protem. 1 U ( endotoxin unit)) T his suggested that MBP- or toxin A repeat-containing lusions with the botulinal ( f ragment should also contain high levels ot endotoxin Accoidinglv icmov al oi endotoxin fiom af finitv -purified pMal-c and pMBot protem prepaiations was attempted as lollows
Samples of pMal-c and pMBot protein were depvrogenated with polvmv xin to determine it the endotoxin could be easilv removed The tollovving amount ot protein vvas tieated 29 ml at 4 8 OD^/ml for pMal-c and 19 mis at I 44 OD,KI),ml lor pMBot I he protem samples were dialvzed extensivelv against PBS and mixed in a 50 ml tube (Falcon) w ith 0 5 ml PBS-equilibrated pol mvxin B (Affi-Prep Pol mvxin BioRad) T he samples weie allowed to mix bv rotating the tubes overnight at 4°C The polv mvxin was pelleted bv centrifugation lor 30 mm in a bench top centiifuge at maximum speed ( approximatelv 2000 \ g) and the supernatant was icmoved Fhe recovered protem (in the supernatant) was quantified by ODΛ (). and the endotoxin activity was assaveu bv I Al In both cases onlv approximatelv 1/3 of the input protein was recovered and the polv myxm-treated protein retained significant endotoxin contamination (approximatelv 7000 L i ning ol pMBot) The depyrogenation experiment was repeated using an independently purified pMal-c protein preparation and similar results were obtained Fiom these studies it was concluded that significant levels of endotoxin copuπfies with these MBP fusion proteins using the arm lose resin Furthermore, this endotoxin cannot be easilv removed by polvmvxin tieatment
T hese results suggest that the presence of the MBP sequences on the fusion protein complicated the removal ol endotoxin liom preparations ot the pMBot protem
b) Generation Of C. botulinum C Fragment Protein Free Of The MBP
It was demonstiated that the pMBot fusion protein could not be easilv purified from contaminating endotoxin in section a) above The abilitv to produce a pyrogen-fiee (e . endotoxin-ti ee) preparation of soluble botulinal C fragment protein free of the MBP tag was next inv estigated 1 he pMBot expression construct was designed to facilitate purification ol the botulinal ( Iragment from the MBP tag by cleavage of the tusion protem bv utilizing an engineered Factoi λa cleavage site present between the MBP and the botulinal C Iragment I he I ucloi Xa cleav age was performed as follows
1 actor Xa ( New I ngland Biolabs) was added to the pMBot protein ( using a 0 I - I 0% I actoi Xa/pMBot piotein ratio) in a variety ot buffer conditions [e g . PBS-NaCT (PBS containing 0 5 M NaC l ). PBS-NaCl containing 0 2% Tween 20. PBS. PBS containing 0 2%
I een 20. PBS-C (PBS containing 2 mM CaCl,). PBS-C containing either 0 1 or 0 5 % I ween 20. PBS-C containing either 0 i or 0 5% NP-40. PBS-C containing either 0 1 oi 0 5% Futon X- 100 PBS-C containing 0 1 % sodium deoxvcholate. PBS-C containing 0 1 % SDS] I he 1 actoi \a digestions wei e incubated tor 12-72 hrs at room temperature [ he extent ot cleavage was assessed by Western blot or Coomassie blue staining ol proteins following electrophoresis on denaturing SDS-PAGE gels, as described in Example 22 leav age l eactions (and control samples of uncleaved pMBot protein) were centrifuged toi 2 mm in a microfuge to lemove insoluble protein pnor to loading the samples on the gel I he 1 actor Xa tieated samples were compared with uncleav ed. unccntπ f uged pMBot samples on the same gel T he results of this analysis is summarized below
1 ) Most (about 90%) pMBot protein could be icmov ed bv centrifugation. ev en when uncleav ed control samples vvete utilized This indicated that the pMBot fusion protem was not fullv soluble (; e . it exists as a suspension rather than as a solution) [This result was consistent with the observation that most affinity-purified pMBot protein precipitates after long term storage (>2 weeks) at 4°C. Additionally, the majority ( i. e.. 75%) of induced pMBot protein remains in the pellet after sonication and clarification of the induced E. coli. Resuspcnsion of these insoluble pellets in PBS followed by sonication results in partial solubilization of the insoluble pMBot protein in the pellets. )
2) The portion of pMBot protein that is fully in solution (about 10% of pMBot protein) is completely cleaved by Factor Xa. but the cleaved ( released) botulinal C fragment is relatively insoluble such that only the cleaved MBP remains ful ly in solution.
3 ) None of the above reaction conditions enhanced solubility without also reducing effective cleavage. Conditions that effectively solubilized the cleaved botulinal C fragment were not identified.
4) 'Fhe use of 0. 1 % SDS in the buffer used for Factor Xa cleavage enhanced the solubility of the pMBot protein ( all of pMBot protein was soluble). However, the presence of the SDS prevented an cleavage of the fusion protein with Factor Xa. 5 ) Analysis of pelleted protein from the cleavage reactions indicated that both full length pMBot ( i. e.. uncleaved) and cleaved botulinal C fragment protein precipitated during incubation.
T hese results demonstrate that purification of soluble botulinal C fi agment protein after cleavage of the pMBot fusion protein is complicated by the insolubi lity of both the pMBot protein and the cleaved botulinal C fragment protein.
c) Expression Of C. botulinum C Fragment Using Various
Expression Vectors
In order to determine if the solubility of the botulinal C fragment was enhanced by expressing the C fragment protein as a native protein, an N-terminal His-tagged protein or as a fusion w ith glutathione-S-transferase (GST), alternative expression plasmids were constructed. These expression constructs were generated utilizing the methodologies described in Example 22. Figure 27 provides a schematic representation of the vectors described below . In Figure 27. the following abbreviations are used. pP refers to the pET23 v ector. pHIS refers to the pETHisa vector. pBlue refers to the pBluescript vector. pM refers to the pMAL-c vector and pG refers to the pGEXj'F vector (described in Example 1 1 ). T he solid black lines represent C hoiulinum C fragment gene sequences: the solid black ovals represent the MBP: the hatched ovals represent GST: "FIHHHH" represents the poly-histidine tag. In Figure 27. when the name for a restriction enzyme appears inside parenthesis, this indicates that the restriction site was destioved during construction An asterisk appearing with the name foi a restriction enzyme indicates that this restriction site was recreated at a cloning lunction
i) Construction Of pPBot
In oi ei to express the C hoiulinum C fiagment as a native (/ e non-iused) protein the pPBot plasmid (shown schematicallv in Figure 27) was constructed as follows Ihe C liagmeni sequences present in pAlterBot (Example 22) were removed b digestion ot pΛlteiBot with iXcol and Hindlll The \collIIιndll] C tiagment mseit was ligated to pLlllisa veetoi (described 1 xample 18b) which vvas digested with \col and Hindlll This ligation cieates an expression construct in which the Λt l-encodcd methionine of the botuhnal C iragment is ihe initiator codon and directs expression ot the native botuhnal C fiagment Ihe ligation products vvete used to uanslorm competent BI 21(1)13)pl vsS cells (Novagen) Recombinant clones weie identified bv restriction mapping
II) (instruction Of pHisBot
In oidei to expiess the C hoiulinum C fiagment containing a polv-histidme tag at the ammo-terminus ot the recombinant protein, the pHisBot plasmid (shown schematicallv in 1 igure 27) vvas constiucted as follows Ihe X toll Hindlll botuhnal C Iragment insert trom pΛlteibot was ligated into the pETIlisa vector which vvas digested with Mel and Hind]]] Ihe \col (on the C fiagment insert) and Xhel (on the pi THisa veetoi) sues were filled m using the klenow tiagment prior to ligation. these sites weie then blunt end ligated (the Xd site vvas icgeneraled at the clone |unctιoπ as predicted) Ihe ligation pioducts were used to Hailstorm competent BL21(DEi)pl vsS cells and recombinant clones weie identified bv icstiiction mapping
Ihe resulting pHisBot clone expiesses the botulinal C liagment protein with a hislidme-tagged N-terminal extension having the following sequence MetGlvHisI lis HisHisl lisllisHisHisHisHisSeiSerC.lv HislleGluGlvAruHisMetAla. (SEQ ID NO 24) the amino acids encoded bv the botuhnal C Iragment gene are underlined and the vector encoded amino acids aie piesented in plain tvpe The nucleotide sequence present in the pi THisa veetoi which encodes the pHisBot lusion protem is listed in SEQ ID NO 25 The ammo acid sequence of the pHisBot protem is listed in SEQ ID N 26 iii) Construction Of pGBot
The botulinal C fragment protein was expressed as a lusion with the glutathione-S- transferase protein by constructing the pGBot plasmid (shown schematically in Figure 27) This expression construct was created by cloning the oil/Sall C fragment insert present in pBIueBot (Example 22) into the pGEX3T vector which vvas digested with Smal and Xhol
The .WI site ( present on the botulinal fragment) was made blunt prior to ligation using the Klenow fragment. Fhe ligation products were used to transform competent BL21 cells.
Each of the above expression constructs were tested by restriction digestion to confirm the integrity of the constructs I arge scale ( I liter) cultures of pPBot [BL21 ( DE3 )pLysS host], pHisBot
[ BL21 ( DE3 )pL ysS host] and pGBot ( BL21 host) were grown m 2X YT medium and induced ( using I PTG to 0 8- 1 .0 mM) for 3 hrs as described in Example 22 otal, soluble and msoluble protein preparations were prepared from 1 ml aliquots ot each large scale culture I Williams ei al ( 1 994 ). supra] and analyzed bv SDS-PAGF No obv ious induced band was detectable m the pPBot or pHisBot samples by Coomassie staining, while a prominent insoluble band of the anticipated MW was detected in the pGBot sample Soluble ly sates ol the pGBot large scale ( resuspended in PBS) or pl iisBol lai ge scale | esuspended in Novagen I X binding buffer ( 5 mM imidazoie. 0 5 M NaCl. 20 mM I ns-HCl. pl l 7 9) | cultures were prepared and used to affinity purify soluble affinity-tagged protem as follows I he pCiBol lysate was affinitv purified on a glutathio e-agarose resin ( Pharmacia ) exactly as descπbed in Smith and Corcoran [Current Protocols in Molecular Biology . Supplement 28 ( 1994). pp 16 7 1 - 16 7 7|. T he pHisBot protein vvas purified on the His-Bind lesm ( Nov agen ) utilizing the His-bmd buffer kit (Novagen ) exacllv as described bv manufacturer Samples from the purification of both the pGBot and pHisBot proteins ( including uninduced. induced, total, soluble, and affinity-purified eluted protein ) were resolved on SDS- PAGE gels I ollowing electrophoresis. proteins were anal ed bv C oomassie staining oi bv Western blot detection utilizing a chicken antt-C botulinum I vpe Λ toxoid antibody (as described in Example 22). 1 hese studies showed that the pGBot protein was almost entirely insoluble under the utilized conditions, while the plTisBot protein was soluble. Affinity purification of the pHisBot protem on this first attempt vvas inefficient, both m terms of yield ( most of the
.58 immunoreactive botulinal protein did not bind to the Mis-bind resin) and purity (the botulinal protein vvas estimated to comprise approximately 20% of the total eluted protein).
d) Purification Of Soluble C. botulinum C Fragment Protein Substantially Free Of Endotoxin Contamination
T he above studies showed that the pHisBot protein was expressed in E. coh as a soluble protein. However, the affinity purification of this protein on the His-bind resin vvas v ery inefficient. In order to improve the affinity purification of the soluble pHisBot protein ( in terms of both yield and purity), an alternative poly-histidine binding affinity resin (Ni- NTΛ resin: Qiagen) vvas utilized. The Ni-NTA resin was reported to have a superior binding affinit ( K r I x 1 0 "' ' at pH 8.0: Qiagen user manual) relative to the His-bind resin.
■\ soluble ly sate ( in Novagen I X binding buffer) from an induced 1 liter 2X Y culture w as prepared as described above. Briefly, the culture of pHisBot [BI2 1 ( DE3 )pLysS host ] w as grow n at 37°C to an ODw„, of 0.7 in 1 liter of 2X YT medium containing 100 μg/ml ampiciilin. 34 μg/ml chloramphenicol and 0.2% glucose. Protein expression was induced by the addition of IPTG lo 1 mM. Three hours after the addition of the IPTG. the cells were cooled for 1 5 min in a ice water bath and then centrifuged 10 mm at 5000 rpm in a JA M) rotor ( Beckman ) at 4°C. The pellets were resuspended in a total volume of 40 mis Novagen I X binding buffer ( 5 mM imidazoie. 0.5 M NaCl. 20 mM Tris-HCl. pl l 7.9). transferred to two 35 mi Oakridge tubes and frozen at -70°C for at least 1 hr. The tubes were thawed and the cells were lysed by sonication (4 X 20 second bursts using a Branson Sonillcr 450 w ith a power setting of 6-7) on ice. The suspension was clari fied by centrifugation for 20 min at 9.000 rpm ( 10.000 x g) in a JA- 1 7 rotor ( Beckman ).
The soluble lysate was brought to 0. 1 % NP40 and then was batch absorbed to 7 mi oϊ a 1 : 1 slurry of Ni-NTA resimbinding buffer by stirring for 1 hr at 4°C. Fhe slurry was poured into a column having an internal diameter of 1 or 2.5 cm ( BioRad). T he column was then washed sequentially with 15 mis of Novagen I X binding buffer containing 0. 1 % NP40. 1 5 ml of Novagen I X binding buffer. 15 ml wash buffer (60 mM imidazoie. 0.5 M NaCl. 20 mM Tris-HCl. pH 7.9) and 15 ml NaHPO, wash buffer ( 50 mM NaHPO,. pH 7.0. 0.3 M NaCl. 10 % glycerol ). The bound protein was eluted by protonation of the resin using elution buffer ( 50 mM NaHPO.,. pH 4.0. 0.3 M NaCl. 10 % glycerol). T he eluted protein was stored at 4°C. Samples of total, soluble and eluted protein were resolved by SDS-PAGE. Protein samples were prepared for electrophoresis as described in Example 22b. Duplicate gels were stained with Coomassie blue to visualize the resolved proteins and C hoiulinum type A toxin- reactive protein was detected by Western blot analysis as described in Example 22b. A representative Coomassie stained gel is shown in Figure 28. In Figure 28. the following samples were loaded on the 12.5% acrylamide gel. Fanes 1 -4 contain respectively total protein, soluble protein, soluble protein present in the now-through of the Ni-NTA column and affinity-purified pHisBot protein ( i. e.. protein released from the Ni-NTA resin by protonation ). Lane 5 contains high moiecular weight protein markers ( BioRad ). Fhe purification of pHisBot protein resulted in a yield of 7 mg of affinity purified protein from a 1 liter starting culture of BL21 (DE3)pLysS cells harboring the pHisBot plasmid. The yield of purified pHisBot protein represented approximatelv 0.4% of the total soluble protein in the induced culture. Analysis of the purified pHisBot protein bv SDS- PAGE revealed that at least 90-95% of the protein vvas present as a single band ( Figure 28) of the predicted MW (50 kD). T his 50 kD protein band was immunoreacti e w ith anti-C. hoiulinum t pe A toxin antibodies. T he extinction coefficient of the protein preparation vvas determined to be 1 .4 (using the Pierce BCA assay ) or 1 .45 ( using the Fow r assay ) OD x„ per 1 mg/ml solution.
Samples oϊ pH neutralized eluted pHisBot protein were resolved on a K.B 803 HPLC column ( Shodex ). Although His-tagged proteins are retained b this sizing column ( perhaps due to the inherent metal binding ability of the proteins), the relative mobilit of the pHisBot protein was consistent w ith that expected for a non-aggregated protein in solution. Mosl of the induced pHisBot protein was determined to be soluble under the growih and solubilization conditions utilized above (i. e.. greater than 90% of the pHisBot protein was found to be soluble as judged by comparison of the levels of pFlisBot protein seen in total and soluble protein samples prepared from BL21 (DE3)pLysS cells containing the pHisBot plasmid). SDS-PAGE analysis of samples obtained after centrifugation. extended storage at -20°C. and at least 2 cycles of freezing and thawing detected no protein loss ( due to precipitation), indicating that the pHisBot protein is soluble in the elution buffer ( i. e.. 50 mM Nal lPO ,. pll 4.0. 0.3 M NaCl. 10 % glycerol).
Determination of endotoxin contamination in the affinity purified pHisBot preparation (after pH neutralization) using the I .ΛL assay (Associates of Cape Cod) detected no significant endotoxin contamination. T he assay was performed using the endpoint chromogenic method (without diazo-coupling) according to the manufacturer's instructions. This method can detect concentrations of endotoxin greater than or equal to 0.03 EU/ml (FT ' refers to endotoxin units). The LAL assay was run using 0.5 ml of a solution comprising 0.5 mg pHisBot protein in 50 mM NaHPO,. pl l 7.0. 0.3 M NaCl. 10 % glycerol: 30-60 EU were detected in the 0.5 ml sample. Therefore, the affinity purified pHisBot preparation contains
60- 1 20 EU/mg of protein. FDA Guidelines for the administration of parenterai drugs require that a composition to be administered to a human contain less than 5 EU/kg body weight ( Flic av erage human body weight is 70 kg: therefore up to 349 El ) units can be delivered in a parental dose. ). Because very small amount of protem are administered in a vaccine preparation (generally in the range of 1 0-500 μg of protein), administration of affinity purified pl iisBol containing 60- 120 EU/mg protem would result in delivery ol only a small percentage of the permissible endotoxin load. For example, administration ot 10-500 μg of purified pHisBot to a 70 kg human, where the protem preparation contains 60 EU/mg protem. results in the introduction ot only 0.6 to 30 EU (/ c. 0.2 to 8 6% of the maximum allowable endotoxin burden per parenterai dose ( less than 5 EU/kg body weight)].
T he above results demonstrate that endotoxin ( EPS) does not copurily w ith the pHisBot protein using the above purification scheme Preparations of recombinantly produced pHisBot protein containing lower levels of endotoxin ( less than or equal to 2 FU/ mg recombinant protein ) may be produced by washing the Ni-NT A column w ith wash buffer until the ()D.M, returns to baseline levels ( / e.. until no more UV-absorbing material comes oft of the column ).
I he above results illustrate a method for the production and purification ot soluble, botulinal C fragment protein substantially free of endotoxin.
EXAMPLE 25
Optimization Of The Expression And Purification Of pHisBot Protein
The results shown in Example 24d demonstrated that the pHisBot protein is an excellent candidate for use as a v accine as it could be produced as a soluble protein in /;' c oli and could be purified free of pv rogen activity. In order to optimize the expression and purification of the pHisBot protein, a variety of growth and purification conditions were tested a) Growth Parameters i) Host Strains
The influence of the host strain utilized upon the production of soluble pHisBot protein was investigated A large scale purification ot pHisBot was performed [as described in Example 24d above] using the BL2KDE3) host (Novagen) rather than the
BL21(DE3)pLysS host. Fhe deletion of the pl.ysS plasmid in the BL21(DE3) host yielded higher levels oϊ expression due to de-repression of the plasmicFs T7-lac promoter However, the yield of affinity-purified soluble recombinant protein was veiv low (approximatelv 600 μg/ 11 l i culture) when purified under conditions identical to those described m E.xample 24d above This result was due to the fact that expression in the BL2KDE3) host vielded very high level expression ol the pHisBot protem as insoluble inclusion bodies as shown by SDS- PAGL analysis of protein prepared from induced BL2I(DE3) cultures (Figuie 29. lanes 1-7. desciibed below) These results demonstrate that the pHisBot protein is not inherently toxic to L coli cells and can be expressed to high levels using the appropriate promotei/host combination.
I igure 29 shows a Coomassie blue stained SDS-PAGF gel (125% acrylamide) onto which extracts prepared from BL.21(DF!3) cells containing the pHisBot plasmid were loaded Each lane was loaded with 25 μl protem sample mixed with 25 μl of 2X SDS sample buffei The samples were handled as described in Example 22b Ihe tollovving samples weie applied to the gel Lanes 1-7 contain protein isolated from the BL2KDF3) host I anes 8-14 contain proteins isolated From the BL2KDI 3)pLysS host lotal protein vvas loaded in lanes 1.2.4 6.8. 10 and 12 Soluble protem was loaded in Lanes 3.5.7.9. I I and P Lane 1 contains protein t om uninduced host cells. Lanes 2-13 contain protein Irom host cells induced for 3 hours IPTG vvas added to a final concentration of 01 mM (Lanes 6-7).0 mM (Lanes 4-5) or 10 mM (Lanes 2.3.8-13) The cultures were grown in LB broth (I anes 8-9).2X Yl broth (Lanes 10-11) or terrific broth (Lanes 1-7.12-13) The pHisBot piotein seen in I anes 3.5 and 7 is msoluble protein which spilled over from Lanes 2.4 and 6. lespectivelv High molecular weight protein markers (BioRad) were loaded in Lane 14
Λ variety ot expression conditions were tested to deteimme if the BI 21(DF3) host could be utilized to express soluble pHisBot protein at suitably high levels (i c . about 10 mg/ml) The conditions altered were temperature (giovvth at 37 oi 30°C). culture medium <2X YT. FB oi lerπfic broth) and lnducer levels (01.03 oi 10 mM IPTG) All combinations of these variables were tested and the induction levels and solubihtv vvas then assessed bv SDS-PAGE analysis ot total and soluble extracts [prepared from 1 ml samples as described m Williams et al . (1994). supra]
All cultures weie grown in 15 ml tubes (Falcon #2057) All culture medium was prewarmed overnight at the appropriate temperature and were supplemented with 100 μg/ml ampiciilin and 02% glucose Terrific broth contains 12 g/1 bacto-tryptone.24 g/1 bacto-yeast exti ct and 100 ml/I of a solution comprising 017 M HTOj 072 M K,HPO4 Cultures were giown in a incubator on a lotating wheel (to ensure aeration) to an ODWK1 of approximately 04 and induced by the addition of IPTG In all cases high level expression ol insoluble pFlisBot piotein was observed regardless ot temperature medium oi lnducer eoncentiation
Ihe eltect ol vaiving the concentration of IP 1 G upon 2X YT cultures grown at 23°C vvas then investigated IPFG was added to a final concentration of eithei 1 mM 01 mM
005 mM oi 001 mM \t this temperatuie similar levels of pHis Bot protein was induced in the presence ol eithei I or 01 mM IPTG these levels of expression was lower than that observed at lughei temperatures Induced protein levels were reduced at 005 mM IPTCJ and absent at 001 mM IPTG (relative to 10 and 01 mM IPTG inductions at 23°C) However no conditions were observed in which the induced pHisBot protem was soluble m this host
1 hus although expression levels aie superior in the BL2KDI 3) host (as compared to the BL2KD13)pl vsS host) conditions that facilitate the production of soluble protein in this host could not be identified
I hese icsults demonstrate that production of soluble pHisBot protein was achieved using the BI 21 ( DT )pl vsS host in conμinction with the T7-lac piomotei
ii) Lffect Of Varying Temperature, Medium And IPTG Concentration And Length Of Induction
The effect growing the host cells in various mediums upon the expression of iecombinant botulinal protein Irom the pHisBot expression construct [in the BL2KDI 3)pl vsS host] was investigated BL21(Dr3)pLysS cells containing the pHisBot plasmid weie giown in eithei 1 B.2X YI or Terrific broth at 37°C Fhe cells weie induced using I mM IPTCJ for a 3 hi induction period Expression of pFlisBot protein was found to be the highest when the cells eie grown in 2X YT broth (see figure 29 lanes 8-13)
The cells were then giown at 30°C in 2X YT broth and the eoncentiation of IITG was varied fiom 10 03 ot 01 mM and the length ot induction was either 3 or 5 houis Expression of pHisBot protein was similar at all 3 inducer concentrations utilized and the levels of induced protein were higher after a 5 hr induction as compared to a 3 hr induction.
Using the conditions found to be optimal for the expression of pHisBot protein, a large scale culture was grown in order to provide sufficient material for a large scale purification of the pHisBot protein. Three 1 liter cultures were grown in 2X YT medium containing 100 μg/ml ampiciilin. 34 μg/ml chloramphenicol and 0.2% glucose. The cultures were grown at 30°C and were induced with 1 .0 mM IPTG for a 5 hr period. The cultures were harvested and a soluble lysate were prepared as described in Example 1 8. A large scale purification vvas performed as described in Example 24d with the exception that except the soluble lysate vvas batch absorbed for 3 hours rather than for 1 hour. T he final yield was 1 3 mg pHisBot protein/liter culture. T he pHisBot protein represented 0.75% of the total soluble protein.
The above results demonstrate growth conditions under which soluble pHisBot protein is produced ( i. e.. use of the BL21 ( DE3 )pLysS host. 2X Y medium. 30°C. LO mM I PTG for 5 hours).
b) Optimization Of Purification Parameters l or optimization of purification conditions, large scale cultures (3 X 1 liter) were grown at 30°C and induced with I mM I PTG for 5 hours as described above. T he cultures were pooled, distributed to centrifuge bottles, cooled and pelleted as described in Example 24d. T he cell pellets were frozen at -7()°C until used. Each cell pellet represented 1 /3 of a liter starting culture and individual bottles were utilized for each optimization experiment described below . This standardized the input bacteria u.sed for each experiment, such that the yields of affinity purified pHisBot protein could be compared between different optimization experiments.
i) Binding Specificity (pll Protonation)
A lysate of pHisBot culture was prepared in PBS ( pH 8.0) and applied to a 3 ml Ni- NTA column equilibrated in PBS (pl l 8.0) using a How rate of 0.2 ml/min ( 3-4 column v oluines/hr) using an Econo chromatography system (BioRad ). I he column was washed with PBS (pl l 8.0) until the absorbance (OD s„) of the elute as at baseline levels. he (low rate vvas then increased to 2 ml/min and the column was equilibrated in PBS ( pl l 7.0). A pl l gradient ( pl l 7.0 to 4.0 in PBS) was applied in order to elute the bound pHisBot protein from the column. Fractions were collected and aliquots were resolved on SDS-PAGE gels. T Tie PAGE gels were subjected to Western blotting and the pHisBot protein was detected using a chicken anti-C hoiulinum l ype A toxoid antibodv as described in Example 22
I rom the Western blot analysis it was determined that the pHisBot protem begins to elute f iom the Ni-NTA column at pH 6 0 t his is consistent with the predicted elution ol a His-tagged protem monomer at pH 5 9
I hese results demonstrate that the pH at which the pHisBot piotem is protonated ( l eleased ) trom Ni-N I Λ resin in PBS buffei is pH 6 0
ii) Binding Specificity (Imidazoie Competition)
In older to define purification conditions under which the native L coli proteins could be remov ed liom the Ni-NT A column w hile leaving the pHisBot protein bound to the column the following experiment was performed A Iv sate ol pHisBot cultuie was piepared in 50 m NaHPO, 0 5 M NaC 1 8 mM imidazoie ( pl l 7 0) I his Iv sate was applied to a 3 ml Ni- I \ column equilibrated in 50 mM NaHPO., 0 5 M NaCl (pH 7 0) using an Econo chi omatographv sv stem ( BioRad) A flow rate of 0 2 ml/min (3-4 coiumn volumes/hr) was utilized T he column was washed w ith 50 mM NaHPO.,. 0 5 M NaC l (pH 7 0) until the absoi bance ol the elute returned to baseline I he flow late was then increased to 2 ml/mm
I he column was eluted using an imidazoie step gi adient | ιn 50 mM NaHPO,. 0 5 M NaCT ( pl l 7 0)| F lution steps were 20 mM. 40 mM. 60 mM 80 mM. 100 mM. 200 mM. 1 0 M imidazoie. lollowed bv a wash using 0 1 mM EDTA (to strip the nickel li om the column and l emov e remaining piotein) In each step, the wash vvas continued until the OD „, l etui ne l to baseline I l actions were resolved on SDS-PAGT gels \\ estern blotted, and pHisBot protein detected using a chicken anti-C botulinum Ty pe A toxoid antibodv as desci ibed in I xample 22 Duplicate gels were stained w ith C oomassie blue to detect eluted piotem in each fraction
I he results ot the PAGE analysis showed that most of the non-specifically binding bacterial protein was removed by the 20 mM lmidiazole wash, with the remaining bacterial proteins being l emoved in the 40 and 60 mM imidazoie washes I he pHisBot pi otein began to elute at 100 mM imidazoie and was quantitatively eluted in 200 mM imidazoie
I hese results piecisely defined the window of imidazoie wash stπngencv that optimallv icmov es L c oli proteins f rom the column while specificallv retaining the pHisBot protem in this buf fer Fhese results provided conditions under which the pHisBot protein can be purified Iree ot contaminating host proteins iii) Purification Buffers And Optimized Purification Protocols
A variety ot purification parameters were tested during the development ot an optimized protocol for batch purification ol soluble pHisBot protein I he icsults of these analyses aie summarized below
Batch purifications were performed (as described m I xample 24d) using several buffers to determine il alternative buffers could be utilized tor binding ot the pFlisBot protein to the Ni-N TA column It was determined that quantitativ e binding ot pHisBot protein to the Ni-NTA lesin was achieved in either 1 πs-HCl (pl l 7 9) or NaHPO., (pl l 8 0) buffers Binding of the pHisBot protein in NaHPOj buf fer was not inhibited using 5 mM. 8 mM oi 60 mM imidazoie Quantitative elution of bound pI lisBot protein was obtained in buffers containing 50 mM NaHPO,. 0 3 M NaCl (pH 3 5-4 0). ith oi without 10% gly cerol l lowev ei . quantitation of soluble af finity purified pHisBot pi otem belore and alter a f ieeze thaw ( follow ing several weeks storage ol the affinitv purified elute at -20°C ) i ev ealed that 94% of the protein w as recovered using the glycerol-containing buf fei . but only 68% ol the protein was lecovered when the buffer lacking glycerol was employed I bis demonstrates that gly cei ol enhanced the solubility ot the pHisBot protein in this low pl l butler when the eluted protem vvas stoied at freezing temperatures (e g . -20°C) Neutralization ot pH bv addition ol NalLPO, buffer did not result in obvious protem precipitation It vvas determined that quantitative binding ol pHisBot protem using the batch loi mat occuπcd altei hrs ( F igure 30). but not alter 1 hr ot binding at 4°C (the i csin was stu red during binding ) Figure 30 depicts a Coomaisse blue stained SDS-PΛGI gel ( 7 5% aciy lamide) containing samples oi proteins isolated during tlie purification ot pHisBot protein liom ly sate pi epared from the BL21 (DE3)pLysS host Each lane was loaded w ith 5 μl of protein sample mixed with 5 μl ot 2X sample buffer and processed as described in Example
22b Lane 1 contains high molecular weight protein markers ( BioRad) I anes 2 and 3 contain protein eluted from the Ni-NTA i csin Lane 4 contains soluble pi otein attei a 3 lit batch incubation w ith the Ni-NT A resin Lanes 5 and 6 contain soluble and lotal protem. respectiv ely 1 igure 30 demonstrates that the pHisBot protein is completelv soluble [compare Lanes 5 and 6 which show that a similar amount ol the 50 k pHisBot piote is seen in both, if a substantial amount (greater than 20%) of the pHisBot protein were partially msoluble in the host cell, more pHisBot protein would be seen in lane 6 ( total protem) as compared to lane 5 (soluble protem) | figuie 30 also demonstrates that the pHisBot protein is completelv removed from the lysate after batch absorption with the Ni-NTA resin toi 3 hours (compare Lanes 4 and 5)
T he reported high af finitv interaction of the Ni-NT A resin w ith His-tagged proteins (Kd=- I x 10 ' at pH 8 0) suggested that it should be possible to manipulate the resin-protein 5 complexes without significant release of the bound protem Indeed it was determined that after the recombinant protein was bound to the Ni-NT Λ resin, the resm-pHisBot protein complex vvas lughlv stable and remained bound following repeated rounds of centrifugation of ihe l esin f oi 2 nun at 1600 x g When this centrifugation step was performed in a 50 ml tube ( Falcon ) a tight resin pellet formed This allowed the removal ol spent soluble Iv sate bv
10 pouring of f the supernatant followed bv resuspension of the pellet in wash butter I urther washes can be perf ormed bv centrif ugation The abihtv to perform additional w ashes permits the dev elopment of protocols tor batch absorption ol large v olumes ot Ivsate w ith remov al of the Iv sate being pet formed simplv bv centrifugation following binding ot the iecombinant piotem to the l esin
I 5 A simplified integrated purification protocol was dev eloped as lollow s \ soluble
Iv sate vvas made bv resuspending the induced cell pellet in binding buffer [ 50 mM NaHPO, 0 5 \1 NaC l 60 mM imidazoie ( pH 8 0)| sonicating 4 x 20 sec and centrifuging lor 20 mm at I 0 000 \ g NP-40 was added to 0 l % and Ni-NT A lesin (equihbiated in binding buffer) vvas added Light millihtei s ol a l l slurrv ( lesin binding buffer) vvas used per liter ot 0 starting cultui e I he mixture was stirred tor 3 hrs at 4°C I he slurrv was poured into a column hav ing a I cm internal diametei ( BioRad) washed w ith binding butler containing 0 LO NP40 then binding buf fer until baseline was established (these steps mav altemativ eiv be pei toi med bv centrifugation ot the icsin resuspension in binding buf fer containing NP40 followed bv centrifugation and resuspension in binding buffer) Imidazoie was removed bv
25 washing the resin with 50 mM NaHPO, 0 3M NaCl (pH 7 0) Protem bound to the resin was eluted using the same butter (50 mM NaHPO.,, 0 3M NaCl) having a reduced pH ( pH 3 5- 4 0)
A pilot purification was performed follow ing this protocol and v ielded 1 mg/htei al finitv -punfied pHisBot The pHisBot protein vvas gieater than 90% pure as estimated bv
30 C oomassie staining ol an SDS-PAGI gel This represents the highest observ ed v icld ot soluble affinitv -purified pHisBot protein and this protocol eliminates the need tor separate imidazole-contaming binding and wash buf fet s In addition to pioviding a simplified and efficient protocol for the affinitv purification of iecombinant pHisBot protein the above results provide a variety of purification conditions under which pHisBot protein can be isolated.
EXAMPLE 26 T he pHisBot Protein Is An Effectiv e Immunogen
I n Example 23 it was demonstrated that neutralizing antibodies are generated in mouse serum after nasal immunization with the pMBot protein. However, the pMBot protein was found to copurify with significant amounts of endotoxin which could not be easily removed. The pHisBot protein, in contrast, could be isolated free of significant endotoxin contamination making pHisBot a superior candidate for vaccine production. To further assess the suitability of pI lisBot as a vaccine, the immunogenicity of the pHisBot protein was determined and a comparison of the relative immunogenicity of pMBot and pHisBot proteins in mice was performed as follows. Two groups of eight BALBc mice were immunized with either pMBot protein or pI lisBot protein using Gerbu GMDP adjuvant (CC Biotech ). pMBot protein ( in PBS containing 1 0 mM maltose) or pHisBot protein ( in 50 mMNaHPO,. 0.3 M NaCl. 1 0% glycerol. pH 4.0) was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received an I P injection of 100 μl antigen/adjuvant mix ( 50 μg antigen plus 1 μg adjuvant) on day 0. Mice w ere boosted as described above with the exception that the route of administration was 1M on day 14 and 28. The mice were bled on day 77 and anti-C hoiulinum Type A toxoid titers w ere determined using serum collected from individual mice in each group ( as described in E.xample 23). The results are show n in Table 4 1 .
T VBI.K 41
Figure imgf000171_0002
I lie prcimimme sample represents the av r g Irom 2 sels ol duplicate welts containinϋ serum Irom a individual mouse iπiuium/cd w ith recombinant S φln lm oe ais
Figure imgf000171_0001
I) (SI U nmi_.cn I Ins ainm-n is immunolOHicalh unrelated lo l hi, minium losm and provides a control serum
U cr.ϋ e ol duplicate wells
T he results shown above in Table 41 demonstrate that both the pMBot and pFlisBot proteins are immunogenic in mice as 100% of the mice ( 8/8) in each group seroconverted from non-immune to immune status. The results also show that the average titer of anti-C. hoiulinum y pe A toxoid IgG is 2-3 fold higher after immunization with the pHisBot protein relativ e to immunization with the pMBot protein. This suggests that the pFlisBot protein may be a superior immunogen to the pMBot protein.
EXAMPLE 27
Immunization With The Recombinant pHisBot Protein Generates Neutralizing Antibodies
T he results shown in Example 26 demonstrated that both the pHisBot and pMBot proteins were capable of inducing high titers of anti-C botulinum type A toxoid-reactive antibodies in immunized hosts. The ability of the immune sera from mice immunized with either the pHisBot or pMBot proteins to neutralize ( ' botulinum type A toxoid in vivo was determined using the mouse neutralization assay described in E.xample 23b. The two groups of eight BAEBc mice immunized with either pMBot protein or pHisBot protein in Example 26 were boosted again one week after the bleeding on day 77 T he boost was performed by mixing pMBot protein (in PBS containing 10 mM maltose) or pHisBot protein ( in 50 mM NaHPΩ4. 0 3 M NaCl. 10% glycerol. pH 4 0) with Gerbu adiuvant as described Example 26 Each mouse received an IP lniection ol 100 μl antigen/adμivant mix ( 50 μg antigen plus 1 μg adjuvant) The mice were bled 6 days after this boost and the serum from mice within a group was pooled Serum trom preimmune mice was also collected (this serum is the same serum described in the footnote lo 1 able 41 ) I he presence of neutralizing antibodies in the pooled or preimmune serum was detected by challenging mice with 5 LD, units of type A toxin mixed w ith 100 μl ot pooled serum The challenge as performed by mixing (per mouse to be miected) 100 μl of serum f rom each pool with 100 μl of purified tvpe A toxin standard ( 50 ED 1 'ml prepared as desci ibed in I xample 23b) and 500 μl of gel-phosphate I he mixtures were incubated tor 30 nun at room temperature with occasional mixing Each ol lour mice weie miected IP with the mixtures (0 7 ml/mouse) I he mice were observed for signs ol botulism lor 72 hours
Mice l eceiving toxin mixed with serum trom mice immunized with either the pHisBot oi pMBot proteins showed no signs of botulism intoxication In contrast, mice receiv ing preimmune serum died m less than 24 hours
These results demonstrate that antibodies capable ot neutralizing ( ' hoiulinum type A toxm are induced when either of the recombinant ( ' botulinum C fi agment proteins pHisBot oi pMBot aie used as immunogens.
EXAMPLE 28
Cloning And Expression Ol The C Fragment ol ( hoiulinum Serotype A l oxin In E co LJtihzing A Native Gene 1 ragment
I n Example 22 above, a synthetic gene was used to express the (_ f ragment ot ( ' hoiulinum serotype A toxin in £ colt I he synthetic gene icplaced non-pieleired (/ e . tare) codons present in the C fragment gene with codons which are prelerred bv / c oli T he sv nthclic gene was generated because it vvas been reported that genes which hav e a high A/T content ( such as most clostridial genes) creates expression difficulties in E c oli and yeast F urthermore. EaPenotierc et al suggested that problems encountered with the stability (non- fusion constructs) and solubility ( MBP fusion constructs) ot the C Iragment ol C botulinum serotype A toxin when expressed in E coh was most likely due to the extreme A/T richness of the native C botulinum serotype A toxin gene sequences ( LaPenotiere. el al supra)
In this example, it was demonstrated that successful expression of the C fragment of ( hoiulinum type A toxin gene in E coh does not require the elimination of rare codons ^ ( i e . there is no need to use a synthetic gene) This example involved a) the cloning of the nativ e C f iagment of the C botulinum serotype A toxin gene and construction ot an expression vector and b) a comparison of the expression and purification v ields of C hoiulinum serotype A C fragments derived from native and synthetic expression vectors
0 a) Cloning Of The Native C Fragment Of The C. botulinum
Serotype A Toxin Gene And Construction Of An Expression Vector f he seiotv pe A toxm gene was cloned from ( ' hoiulinum genomic DNA using PCR amplification Fhe following primer pair was employed 5'-CGCCA I GGCTAG ^ A I TΛ 1 rA I C I ΛCATTTAC-r ( 5" primer. Xcol site underlined. SFQ ID NO 20) and
S -GCAAGCTI CTTGΛCACACTCA I GTAG-3" (V primer. Hindlll site underlined. SEQ ID NO 30) ( botulinum tv pe A strain was obtained from the American I pe ( ulture Collection I Λ K # 19397) and giown under anaerobic conditions in Terrific broth medium High molecular-weight ( botulinum DNA was isolated as described in E.xample 1 1 The integrity 0 and v ield oi genomic DNA was assessed by comparison with a serial dilution of uncut lambda
DNA alter electiophoresis on an agarose gel
I he gene Iragment was cloned by PCR utilizing a proofreading thermostable DNA polv merase ( nativ e Pfu polymerase) PCR amplification was performed using the above primei pan in a 50μl reaction containing l OmM Tris-HCl (pl l 8 3 ). 50mM KG. 1 5mM 5 iVIg F. 20()μM each dNTP. 0 2μM each primer, and 50ng C hoiulinum genomic DNA
Reactions were overlaid with l OOμ! mineral oil, heated to 94 C 4 min. 0 5μl native Pfu polv merase (Stratagene) vvas added, and thirty cycles comprising 94 "C foi I mm. 50° C for 2 mm. 72 °C lor 2 mm were carried out followed by 10 mm at 72°C An aliquot ( 10μl ) ot the reaction mixture was resolved on an agarose gel and the amplified nativ e C fi gment gene 0 was gel purified using the Prep-A-Gene kit (BioRad) and ligated to pCRScπpt v ector DNA
( Stratagene) Recombinant clones were isolated and confirmed bv restriction digestion, using standard i ecombinant molecular biology techniques [Sambiook el al ( 1989). supra] In addition, the sequence ol approximatelv 300 bases located at the 5" end of the C fragment coding region were obtained using standard DNA sequencing methods The sequence obtained was identical to that o the published sequence
An expression vector containing the native C hoiulinum serotype A C f ragment gene was created b\ ligation of the col-Hmdlll fragment containing the C fragment gene from the pCRScπpt clone to /VAt'I-H/wdlll restricted pETHisa vector ( E xample 1 8b) I he /Viol and
Xbe sites weie filled in using the Klenow enzyme prior to ligation. these sites were thus blunt-end ligated together I he resulting construct was termed pHisBotA ( native) pHisBotA
( native) expresses the ( ' hoiulinum serotype A C f ragment w ith a his-tagged N terminal extension which has the following sequence Met(ilvHιsHιsHιsHιsHιsHιsHιsHιsHιsHιsSerSerGlvHιs// G///G7t/lr. HιsMetAla ( SEQ ID
NO 24). where the underlining represents ammo acids encoded bv the C hoiulinum C
Iragment gene (this N terminal extension contains the recognition site toi 1 actorXa protease. show n in italics, which can be employed to removed the poly histdine tract f rom the N- terminus ol the l usion protein) Fhe pHisBot ( native) construct expresses the identical protein as the pHisBot consti uct ( Ex 24c herein alter the pHisBotA) which contains the s nthetic gene
Fhe piedicted DNA sequence encoding the native ( hoiulinum serotv pe Λ C f ragment gene contained w ithin pHisBotA ( native ) is listed in SEQ I NO 3 1 [ the start ot translation
(AT G) is located at nucleotides 108- 1 10 and the stop ot tianslation (TAA) is located at nucleotides 1494- 1496 in SI Q I D NO 3 1 ] and the corresponding ammo acid sequence is listed in SI Q ID NO 26 ( / e . the same amino acid sequence as that produced bv pHisBotA containing sv nthetic gene sequences)
b) Comparison Of The Expression And Purification Yields Of C. botulinum Serotype A C Fragments Derived From Native
And Synthetic Expression Vectors
Recombinant plasmids containing either the native or the s nthetic ( hoiulinum serotv pe A C ti agment genes were transformed into coli strain B121 (DI 3) pLv sS and prote expiession vv as induced in 1 liter shaker flask cultuies l tal protem extracts were isolated, icsolved on SDS-PAGE gels and ( ' botulinum C ti agment protein was identified bv
Western analysis utilizing a chicken antι-( hoiulinum serotv pe A toxoid antiserum as described in 1 xample 22 Briefly. I liter (2XYT + 100 μg/ml ampiciilin and 34 μg/ml chloramphenicol) cultures of bacteria harboring either the pFlisBotA (synthetic) or pHisBotA (native) plasmids in the BI2 K DE3) pl.ysS strain were induced to express recombinant protein by addition of IPTG to I mM. Cultures were grown at 30-32°C. IPTG was added when the cell density reached an
' 5 OD,,|H, 0.5- 1.0 and the induced protein was allowed to accumulate for 3-4 hrs after induction.
The cells were cooled for 1 5 min in a ice water bath and then centrifuged for 10 min at 5000 rpm in a JA 10 rotor ( Beckman ) at 4°C. The cell pellets were resuspended in a total volume of 40 mis I X binding buffer ( 40 mM imidazoie. 0.5 M NaCl. 50 mM NaP0 . pH 8.0). transferred to two 50 ml Oakridge tubes and frozen at -70°C for at least 1 hr. The tubes
10 were then thawed and the cells were lysed by sonication (using four successive 20 second bursts) on ice. Fhe suspension was clarified by centrifugation 20-30 min at 9.000 rpm ( 10.OOOt ) in a JA- 1 7 rotor. The soluble lysate was batch absorbed to 7 ml of a 1 : 1 slurry of NiNTA resimbinding buffer by stirring 2-4 hr at 4°C. The slurrv vvas centrifuged for 1 min at 5 0, i in 50 ml tube ( Falcon ), resuspended in 5 mis binding buffer and poured into a 2.5 cm
1 5 diameter column ( BioRad). The column was attached to a UV monitor ( ISCO) and the column was washed with binding buffer until a baseline vvas established. I midazoie was remov ed by washing with 50mM NaPO4. 0.3 M NaCl. 10% glycerol. pH 7.0 and bound protein was eluted using 50mM NaPO4. 0.3 M NaCl. 10% glycerol. pH 3.5-4.0.
Fhe eluted proteins were stored at 4°C. Samples of total, soluble, and eluted proteins
20 were resolved by SDS-PAGE. Protein samples were prepared for electrophoresis by mixing l μl total ( T ) or soluble ( S ) protein with 4 μl PBS and 5 μl 2X SDS-PAGE sample buffer, or 5 μl eluted ( E ) protein and 5 μl 2X SDS-PAGE sample buffer. Fhe samples were heated to 95 "C for 5 min. then cooled and 5 or 1 0 μls were loaded on 12.5% SDS-PAGE gels. Broad range molecular weight protein markers ( BioRad) were also loaded to allow the MW of the
25 identified fusion proteins to be estimated. After electrophoresis. protein was detected either generally by staining gels with Coomassie blue, or specifically, by blotting to nitrocellulose for Western blot detection of specific immunoreactive protein.
1 or Western blot analysis, the gels were blotted, and protein transfer was confirmed by Ponceau S staining as described in Example 22. After blocking the blots for I hr at room
30 temperature in blocking buffer (PBST and 5% milk). 10 ml of a 1 /500 dilution of an anti-C hoiulinum toxin A IgY PEG prep ( Ex. 3) in blocking buffer vvas added and the blots were incubated for an additional hour at room temperature. The blots were washed and developed using a rabbit anti-chicken alkaline phosphatase conjugate ( Boehringer Mannheim ) as the secondary antibody as descπbed in Ex 22 This analysis detected C botulinum toxin A- teactiv e proteins in the pHisBotA (native and synthetic) protein samples ( corresponding to the predicted full length proteins identified b) Coomassie staining)
A gel containing proteins expressed from the pHisBot and pHisBot (native) constructs during v arious stages of purification and stained with Coomassie blue is shown in I igure 1
In F iguie 3 1. lanes 1 -4 and 9 contain proteins expressed bv the pHisBotA construct (/ e . the s nthetic gene) and lanes 5-8 contain proteins expressed b\ the pHisBotA (native) construct Lanes I and 5 contain total protein extracts, lanes 2 and o contain soluble protem extracts, lanes 3 and 7 contain proteins which flowed through the NiN I A columns lanes 4 8 and 9 contain piotem eluted from the NiN I A columns and lane 10 contains molecular weight markers
The above purification resulted in a yield of 3 mg (native gene) oi 1 1 mg (svnthetic gene ) ol affinity purified protein trom a 1 liter starting culture of which at least 90-95% of the protein was a single band ot the predicted MW (50kd) and immunoreacttv itv tor i ecombinant ( botulinum serotype A C fiagment protein Other than the lev el ol expression no dif lei ence was observed between the native and the sv nthetic gene expression s\ stems
I hese results demonstrate that soluble ( hoiulinum serotv pe A tiagment protein can be expressed in / c oli and purified utilizing either native or sv nthetic gene sequences
EXAMPLE 29
Generation Of Neutralizing Antibodies Using A Recombinant ( hoiulinum Serotv pe A C 1 ragment Protein Containing A Six Residue His- l ag
In Example 27 neutralizing antibodies were generated utilizing the pHisBotA protein. which contains a histidinc-tagged N-terminal extension comprising 10 histidine residues To determine it the generation of neutralizing antibodies is dependent on the presence of this particular his-tag. a protein containing a shorter N-termmal extension (comprising 6 histidine l esidues) was produced and tested for the ability to generate neutralizing antibodies I his example involved a) the cloning and expiession ot the
Figure imgf000176_0001
n) protein and b) the generation and characterization ot hv peπmmune serum a) Cloning And Expression Of The p6HisBotA(syn) Protein
The pόHisBotA(syn) construct was generated as described below; the term "syn" designates the presence of synthetic gene sequences. This construct expresses the C frgament of the C. botulinum serotype A toxin with a histidine-tagged N terminal extension having the following sequence: MetHisHisHisHisHisHisMetAla (SEQ ID NO:32): the amino acids encoded by the botulinal C fragment gene are underlined and the vector encoded amino acids are presented in plain type.
6XHis oligonucleotides [5"-TATGCATCACCATCACCATCA-3' (SEQ ID NO:33 ) and 5'-CATGTGATGGTGATGGTGATGCA-3" (SEQ ID NO:34) were annealed as follows. One microgram of each oligonucleotide was mixed in total of 20 μl I X reaction buffer 2 (NEB) and the mixture vvas heated at 70°C for 5 min and then incubated at 42°C for 5 min. The annealed oligonucleotides were then ligated with gel purified NdellHindlll cleaved pET23b ( T7 promoter ) or pET21 b (T71ac promoter) DNA and the gel purified Ncoll Hindlll C hoiulinum serotype A C fragment synthetic gene fragment derived from pAlterBot ( Ex. 22). Recombinant clones were i.solated and confirmed by restriction digestion. The DNA sequence encoding the 6X his-tagged BotA protein contained within pόHisBotA(syn) is listed in SEQ ID NO:35. T he amino acid sequence of the pόXFIisBotA protein is listed in SEQ ID NO:36. T he resulting recombinant pόXFIisBotA plasmid was transformed into the BE2 KDE3) pLysS strain, and 1 liter cultures were grown, induced and harvested as described in Example 28. His-tagged protein vvas purified as described in Example 28. with the following modifications. T he binding buffer ( BB) contained 5 mM imidazoie rather than 40 mM imidazoie and NP40 as added to the soluble lysate to a final concentration of 0. 1 %. T he bound material was washed on the column with BB until the baseline vvas established, then the column w as w ashed successively with BB+20 mM imidazoie and BB+40 mM imidazoie. The column was eluted as described in Example 28.
I n the case of the pET23-derived expression system, high level expression of insoluble 6FlisBotA protein was induced. The pET21 -derived vector expressed lower levels of soluble protein that bound the NiNTA resin and eluted in the 40 mM imidazoie wash rather than during the low pH eiution. These results (i.e.. low level expression of a soluble protein ) are consistent with the results obtained with pHisBotA protein (Ex. 25): the pHisBotA construct, like the pET2 1 -derived vector, contains the T71ac rather than T7 promoter.
The 6HisBotA protein thus elutes under less stringent conditions than the 10X histidine-containing pHisBot protein ( 100-200 mM imidazoie: Ex. 25) presumably due to the reduction in the length of the his-tag The eluted protein was of the predicted size [i e . shghtlv reduced in comparison to pHisBotA protein]
b) Generation And Characterization Of Hyperimmune Serum Eight BALBc mice wete immunized with purified 6HιsBotA protein using Gerbu
GMDP adjuvant (CC Biotech) T he 40 mM imidazoie elution was mixed with Gerbu adjuv ant and used to immunize mice F ach mouse receiv ed a subcutaneous infection ol 100 μl antιgen/ad]uvant mix ( 12 μg antigen + 1 μg adiuvant) on dav 0 Mice were subcutancouslv boosted as above on dav 14 and bled on day 28 C ontrol mice received pHisBotB protein (prepared as described in Ex 35 below) in Gerbu adiuvant
Antι-( hoiulinum serotype A toxoid titers were determined in serum trom individual mice fi om each group using the ELISA described in E xample 23a with the exception that the initial testing serum dilution was 1 100 in blocking buf fei containing 0 5% I ween 20 lollow ed bv serial 5-fold dilutions into this butter I he results ot the I I ISA demonstrated that seroconv ersion (relative to control mice) occurred in all 8 mice
I he ability ol the antι-( hoiulinum serotype A C fragment antibodies present in serum trom the immunized mice to neutralize native ( hoiulinum tv pe A toxin was tested using the mouse neutiahzation assav described in E xample 23b Fhe amount of neutralizing antibodies present in the serum ot the immunized mice was determined using serum antibodv titrations The v auous sei um dilutions (0 01 nil ) were mixed with 5 I D, , units of ( botulinum tvpe A toxin and the mixtures were injected IP into mice I he neutralizations were perf oi med in duplicate I he mice were then observed for signs ot botulism loi 4 dav s Undiluted serum was lound to protect 100% of the miected mice while the 1 10 diluted serum did not I his corresponds to a neutralization titer ol 0 05-0 5 IU/ml I hese results demonstrate that neutralizing antibodies were induced when the
6HιsBotΛ protem was utilized as the immunogen Furthermore these results demonstiate that sei ocon ersion and the generation of neutralizing antibodies does not depend on the specific N terminal extension present on the recombinant C hoiulinum tv pe A C tiagment proteins EXAMPLE 30
Construction Of Vectors For The Expression Of His-Tagged ( '. botulinum Type A Toxin C Fragment Protein Using the Synthetic Gene
A number of expression vectors were constructed which contained the synthetic C. hoiulinum type A toxin C fragment gene. These constructs vary as to the promoter (T7 or T7lac ) and repressor elements (laclq) present on the plasmid. The T7 promoter is a stronger promoter than is the T71ac promoter. The various constructs provide varying expression levels and varying levels of plasmid stability. This example involved a) the construction of expression vectors containing the synthetic C. botulinum type A C fragment gene and b) the determination of the expression level achieved using plasmids containing either the kanamvcin resistance or the ampiciilin resistance genes in small scale cultures.
a) Construction Of Expression Vectors Containing The Synthetic C. botulinum Type A C Fragment Gene
Expression vectors containing the synthetic C. hoiulinum type A C fragment gene were engineered to utilize the kanamvcin resistance rather than the ampiciilin resistance gene. This vvas done for several reasons including concerns regarding the presence of residual ampiciilin in recombinant protein derived from plasmids containing the ampiciilin resistance gene. In addition, ampiciilin resistant plasmids are more difficult to maintain in culture: the β- lactamase secreted by cells containing ampiciilin resistant plasmids rapidly degrades extracellular ampiciilin. allowing the growth of piasmid-negative cells.
A second altered feature of the expression vectors is the inclusion of laclq gene in the plasmid. This repressor lowers expression from lac regulated promoters (the chromosomally located, lactose regulated T7 polymerase gene and the plasmid located T7lac promoter). This down regulates uninduced protein expression and can enhance the stability of recombinant cell lines. The final alteration to the vectors is the inclusion of either the T7 or F71ac promoters that drive high or moderate level expression of recombinant protein, respectively.
T he expression plasmids were constructed as follows. In all cases, the protein expressed is the pHisBotA(syn) protein previously described, and the only differences between constructs is the alteration of the various regulatory elements described above. i) Construction Of pHisBotA(syn) kan T7Iac
The pHisBotA(syn) kan T71ac construct was made by inserting the Sapl/Xhol fragment containing the ( '. hoiulinum type A C fragment from pHisBotA( syn) into pET24 digested with Sapl/Xhol (Novagen: fragment contains kan gene and origin of replication). The desired construct was selected for kanamvcin resistance and confirmed by restriction digestion.
ii) Construction Of pHisBotA(syn) kan laclq T7lac
Fhe pI IisBotA(syn) kan laclq T71ac construct was made by inserting the A7wI////'wiIII fragment containing the C. botulinum type A C fragment from pHisBotA(syn)kanT71ac into the pF.T24a vector digested with XballHindlll. Fhe resulting construct was confirmed bv restriction digestion.
iii) Construction Of pHisBotA(syn) kan laclq T7
Fhe pHisBotA( syn) kan laclq T7 construct was made by inserting the Λ7?-/I/// ndIII Fragment containing the C. botulinum type A C fragment from pHisBotA(syn ) kan laclq T71ac into Λ7nd/////7dl ll-digested pHisBotB( syn) kan laclq T7 (described in Ex 37c below ) 'Fhe l esulting construct was confirmed by restriction digestion.
b) Determination Of The Expression Level Achieved Using Plasmids Containing Either The Kanamvcin Resistance Or
The Ampiciilin Resistance Genes In Small Scale Cultures
One liter cultures of pHisBotA(syn) kan T71ac/B121 ( DE3)pl.ysS and plTisBotA( syn ) amp T71ac/B121 (DE3)pEysS [this is the previously designated pl IisBotA(syn ) construct ) were grown, induced and his-tagged proteins were purified as described in Example 28. No differences in yield or protein integrity/purity were observed.
These results demonstrate that the antigen induction levels from expression constructs were not affected by the choice of ampiciilin versus kanamvcin antibiotic resistance genes. EXAMPLE 31
Fermentation Of Cells Expressing Recombinant Botulinal Proteins
a) Fermentation Culture Of Cells Expressing Recombinant
5 Botulinal Proteins
I ermentation cultures were grown under the following conditions which were optimized lor growth of the BE21 (DE3) strains containing pFT derived expression vectors An ov ernight 1 liter feeder culture was prepared by inoculating of 1 liter media ( in a 2E shakei flask) with a fresh colony grown on an LB kan plate The feeder culture contained
10 600 nils nitrogen source [20 gm yeast extract (BBL) and 40 gm tryptone ( BBE)/600 mlsj. 200 mis 5X fermentation salts (per liter 48 5 gm K,HP04. 12 gm NaH,P04 «FEO. 5 gm NH4G. 2 5 gm NaCl ). 1 80 mis dl EO. 20 mis 20% glucose. 2 mis 1 M MgS04, 5 mis 0 05M CaCl, and 4 mis ol a 10 mg/ml kanamv cin stock All solutions weie sterilized bv autoclaving. except the kanamv cin stock which was filter sterilized
I ^ Λn aliquot ( 5 ml) ot the feeder culture broth was removed prior to inoculation, and grow n tor 2 days at 37°C as a culture broth sterility control Growth was not observed in this control cultuie in anv ot the fermentations performed
I he inoculated teedei culture was grown for 12- 1 5 hi s (ON ) at 30-37°C Care was taken to prev ent ovcrsaturation of this culture I he saturated feeder culture was added to 10L 0 of lei mentation media in termenter (BiofloIV. New Brunswick Scientific. 1 diso . N l) as follow s I he termenter vvas sterilized 120 mm at 121 °C with dH,0 The sterile water vvas i cmov ed and f ermentation media added as follows 6 liters nitrogen source. 2 liters 5X fermentation salts. 2 liters 2% glucose. 20 mis 1 M MgS0 . 50 mis 0 05 M CaC 2 5-3 5 nils Macol P 400 antitoam (PPG Industries Inc . Gurnee. IE), 40 mis l Omg/ml kanamvcin and
25 1 0 mis ti ace elements ( 8 gm FeSO »7H,O. 2 gm MnS0 «FEO. 2 gm AIG »6H,O. 0 8 gm
CoC I-61 LO 0 4 gm ZnSO,'7H,0. 0 4 gm Na,Mo0 «2FLO. 0 2 gm CuG «2H,O. 0 2 gm NiCF. 0 1 gm H ,BO4/200mls 5 M HG) All solutions were sterilized by autoclaving. except the kanamvcin stock which was filter sterilized Fei mentation media was prcwarmed to 37°C before the addition of the feeder culture
30 After the addition of the feeder culture, the culture was fermented at 37°C. 400 rpm agitation, and 10 I/mιn air sparging Fhe DO-, control vvas set to 20% PID and dissolved en lev els were controlled b} increasing the rate of agitation tiom 400-850 rpm under IX), conttol DO, levels were maintained at greater than ot equal to 20% throughout the entire fermentation. When agitation levels reached 500-600 rpm the temperature was lowered to 30°C to reduce the oxygen consumption rate. Culture growth was continued until endogenous carbon sources were depleted In these fermentations, glucose was depleted first [monitored with a glucose monitoring kit (Sigma)], followed by assimilation of acetate and other acidic carbons [monitored using an acetate test kit (Boehringer Mannheim)] During the assimilation phase, the pH rose from 6 6-6 8 (starting pH) to 7 4-7 5. at which time the bulk of the remaining carbon source was depleted. T his was signaled by a diop in agitation rate (from a maximum of 700-800 rpm) and a rise in DO, levels -»30% T his corresponds to a OD6(HI reading of 18-20/ml At this point a fed batch mode was initiated, in which a feed solution ot 50% glucose was added at a rate of approximately 4 gm glucose/hter/hr I he pH was adjusted to 7.0 b\ the addition of 25% H.P04 (approximatelv 60 mis) Culture growth was continued and reached peak oxygen consumption within the next 3 hrs ot growth (while the remaining residual non-glucose carbon sources were assimilated ) This phase is characterized by a slow increase in pi I. and air sparging was increased to 15L/mιn. to keep the maximum rpm below 850 Once the residual acidic carbon sources are depleted the agitation rate decreases to 650-750 rpm and the pH begins to drop pH control was maintained al 7 0 PID b\ legulated pump addition of a sterile 4M NaOH solution which was consumed at a steady rate for the remainder of the fermentation Growth was continued at 30°C, and the cultures were grown linearly at a growth rate of 4-7 ODM„, units/hr to at least 81 5 ODhlll, units/ml
Figure imgf000182_0001
dry cell weight) without induction Antifoam (a 1 1 dilution with filter sterilized 100% ethanol) was added as necessary throughout the f ermentation to prevent f oaming
During the fed batch mode, glucose was assimilated immediate!} (concentration in media consistently less than 0 1 gm/hter) and acetate was not produced in significant levels by the pE F plasmιd/BL21 (DE3) cell lines tested (approximatelv 1 gm/htei at end of fermentation: this is lower than that observed in harvests from shaker flask cultures utilizing the same strains). T his vvas fortuitous, since high levels ol acetate has been shown to inhibit induction levels in a v ariety of expression systems Fhe above described conditions were found to be highly reproducible between fermentations and utilizing different expression plasmids As a result, glucose and acetate level monitoring were no longer prelormed during fermentation b) Induction Of Fermentation Cultures
Induction with IPTG (250 mg- 10 gms, depending on the expression vector and experiment) was initiated 1 -3 hrs after initiation of the glucose feed (30-50 ODN)( ml). The growth rate after induction was monitored on a hourly basis. Aliquots (5- 10 ml) of cells were harvested at the time of induction, and at hourly intervals post-induction. Optical density readings were determined by measuring the absorbance at 600 nm of 10 μl culture in 990 μl PBS versus a PBS control. The growth rate after induction was found to vary depending on the expression system utilized.
c) Monitoring Of Fermentation Cultures
Fermentation cultures were monitored using the following control assays.
i) Colony Forming Ability
An aliquots of cells were removed from the cultures at each timepoint sampled ( uninduced and at various times after induction) were serially diluted in PBS (dilution 1 = 1 5 μl cells/3 ml PBS. dilution 2 - 1 5 μl of dilution 1/3 ml PBS. dilution 3 - 3 or 6 μl of dilution 2/3mls PBS) and 100 μl of dilution 3 vvas plated on an LB or TSA (trypticase soy agar) plate. The plates were incubated ON at 37°C and then the colonies are counted and scored for macro or micro growih.
ii) Phenotypic Characterization
Colonies growing on LB or TSA plates (above) from uninduced and induced timepoints were replica plated onto LB+kan. EB+chioramphenicol ( for fermentations utilizing FysS or pACYCGro plasmids). LB+kan-M mM IPTG and LB plates, in this order. The plates were grown 6-8 hrs at 37°C and growih was scored on each plate for a minimum of 40-50 well isolated colonies. The percentage of cells retaining the plasmid at time of induction (i.e.. uninduced cultures immediately prior to the addition of IPTG) was determined to be the # colonies LB+Kan (or chloramphenicol) plate/# colonies LB plate X 100%. The percentage of cells w ith mutated pET plasmids was determined to be the ft colonies LB-t-Kan +T PTG plate/# co/onies LB plate X 100%. Colonies on all LB plates were scored morphologically for E. coh phenotype as a contamination control. Morphologically detectable contaminant colonies were not detected in anv fermentation. iii) Recombinant BotA Protein Induction
A total of 10 OOMW units of cells (e g.. 200 μl ol cells at OD6oιr-50/ml) were removed from each timepoint sample to a 1 5 mi microf uge tube and pelleted foi 2 min at maximum rpm in a microf uge The pellets were resuspended in I ml ol 50 mM NaHPO,. 0 5 M NaCl, 5 40mM imidazoie buffer (pH 6 8) containing 1 mg/ml lysozyme I he samples were incubated toi 20 mm at room temperature and stored ON at -70°C Samples were thawed completely at l oom temperature and sonicated 2 X 10 seconds with a Branson Somfier 450 microtip probe at # 3 power setting The samples were centrifuged for 5 nun at maximum rpm in a microf uge.
10 An aliquot (20 μl ) ol the protem samples were removed to 20 μl 2X sample bulfer. betore oi after centrifugation. lor total and soluble protein extracts, respectivelv I he samples w ere heated to 95°C for 5 min. then cooled and 5 or 10 μl were loaded onto 12 5% SDS-PAGF gels High molecular weight protein markers ( BioRad ) w ere also loaded to allow toi estimation ol the MW ol identified fusion proteins Aftei electrophoiesis. pi otein vvas
I s detected either generally by staining gels with C oomassie blue, or specifically , bv blotting onto nitrocellulose (as descπbed in Lx 28) for Western blot detection of specific his-tagged proteins utilizing a NiN I A-alkalme phosphatase coniugate exactlv as desci ibed bv the manuiactuier ( Qiagen)
20 iv) Recombinant Antigen Purification
At the en ot each fermentation run. 1 - 10 liters ol cultuie were harvested trom the lermenter and the baetenal cells were pelleted bv centrifugation at 6000 rpm tor 10 m in a IΛ 10 rotor ( Beckman) The cell pellets were stored frozen at -70°( or utilized immediatel} w ithout f i eezing Cell pellets were resuspended to 1 5-20% weight to v olume in lesuspension
25 buffer (generally 50 mM NaP()4. 0 5 M NaCl. 40mM imidazoie. pH 6 8 ) and U sed utilizing either sonication or high pressuie homogenization.
1 or sonication. the resuspension bufter was supplemented with lvsozvme to 1 mg/ml. and the suspension was incubated lor 20 m at room temp I he sample vvas then liozen ON at -70°O. thawed and sonicated 4 X 20 seconds at microtip maximum to reduce v iscositv
30 1 or homogenization. the cells wete l ed by 2 passes through a homogenizer ( Rannie
Mini-lab tv pe 8 30 IT ) at 600 Bai Cell lysates were clarified bv centrif ugation for 30 mm at 1 0.000 rpm in a JA M) rotor For IDA chromatography. samples were flocculated utilizing polyethyleneimine (PEI) prior to centrifugation. Cell pellets were resuspended in cell resuspension buffer (CRB: 50 mM NaP04, 0.5 M NaCl. 40 mM imidazoie. pH 6.8) to create a 20% cell suspension (wet weight of cells/volume of CRB) and cell lysates were prepared as described above (sonication or homogenization). PEI (a 2% solution in dH,0, pFI 7.5 with HG) was added to the cell lysate a final concentration of 0.2%. and stirred for 20 min at room temperature prior to centrifugation (8.500 rpm in JA 10 rotor for 30 minutes at 4°C). This treatment removed RNA. DNA and cell wall components, resulting in a clarified, low viscosity lysate ("PEI clarified Iv sate"). His-tagged proteins were purified from soluble lysates by metal-cheiate affinity chromatography using either a NiNTA resin (as described in Ex. 28) or an IDA (iminodiacetic acid ) resin as described below.
I DA resin affinity purifications were performed utilizing a low pressure chromaiography system ( ISCO). A 7 ml ( small scale) or 70 ml (large scale) Chelating Sepharose Fast Flow ( Pharmacia) affinity column was poured: in addition, a second guard column was poured and attached in line with the first column ( to capture Ni ions that leached off the affinity column ). The columns were washed with 3 column volumes of dILO. The guard column was then removed and the affinity column vvas washed with 0.3 M NiS04 until resistivity w as established, then with dI LO until the resistivity returned to baseline. The columns were reconnected and equilibrated with cell resuspension buffer ((TIB; 50 mM
NaPO,. 0.5 M NaCl. 40 mM imidazoie. pH 6.8). The clarified sample ( in CRB) was loaded. Flow rates were 5 ml/min for small scale columns and 20 ml/min f or large scale columns. After sample loading, the column was washed with CRB until a baseline established and bound protein vvas eluted with elution buffer (50 mM NaP04. 0.5 M NaCl. 800 mM imidazoie. 20% glycerol. pH 6.8 or 8.0). Protein samples were stored at 4°C or -20°C. T he v ield of eluted protein vvas established by measuring the O:m of the elutions. with a I mg/ml solution of protein assumed to yield an absorbance reading of 2.0.
T he IDA columns may be regenerated and reused multiple times (>10). To regenerate the column, the column was washed with 2-3 column volumes of H,O. then 0.05 M EDTA until all of the blue/green color was removed followed by a wash with dH,0. The IDA columns were sterilized with 0. 1 M NaOFl (using at least 3 column volumes but not more than 50 minutes contact time with column packing material), then washed with 3 column v olumes 0.05 M NaPO ,. pl l 5.0. then dI LO and stored at room temperature in 20 % ethanol. EXAMPLE 32
Construction Of A Folding Chaperone Overexpression System
Co-overe.xpression of the E coh GroEL/GroES folding chaperones in a cell expressing a recombinant foreign protein has been reported to enhance the solubility ot some foreign proteins that are otherwise insoluble when expressed in E colt [(iragerouu et al ( 1992) Proc Natl Acad Sci USA 89 10344] The improvement in solubility is thought to be due to chaperone-mediated binding and unfolding of insoluble denatured proteins, thus allowing multiple attempts for productive refolding of recombinant proteins By ov erexpressmg the chaperones. the unfolding/refolding reaction is d ven by excess chaperone. lesulting, in some cases, in higher yields of soluble protein
In this example, a chaperone overexpression svstem. compatible with pE F vector expression svstems. was constructed to facilitate testing chaperone-mediated solubilization of ( botulinum type A proteins This example involved the cloning ot the Gi ol L T S operon and construction of a pi ysS-based chaperone hypcrexpression system
I he GroEL/GroES operon was PCR amplified and cloned into the pC RScπpt vector as desci ibed in Example 28 T he following primer pair was used 5' -CGC AT ATGΛΛ I A I TCGTCCATTGCATG-3" (SEQ ID NO 37) [5 " primer , start codon ol groFS gene conv erted to iXdel site (underlined)] and 5'-GGAAGCTTGCAGGGCAA7 T 1CATCATG (SEQ ID NO 38) (3' primer, stop codon ot groEL gene italicized, engineered Hindlll site underlined) I ollowrng amplification, the chaperone operon was excised as an \de]l Hindlll Iragment and cloned into pET23b digested with Nde and Hindlll T his constr uction places the G o operon under the control ot the T7 promoter ot the pf 23 v ector I he desired construct was confirmed by restriction digestion T he F7 promoter-Gro operon-T7 terminator expression cassette was then excised as a
Bgl llBsp l (filled) Iragment and cloned into BamHI (compatible with /ii,'/H)/// dIH (filled) cleaved pi vsS plasmid (this removed the T7 lysozyme gene) Fhe icsulting construct was designated pACYCGro. since the plasmid utilizing the pACYC 1 84 origin f i om the plysS plasmid Pi oper construction was confirmed by restriction digestion pACYCGro was transformed into BL21 (DE3). cultuies were grown and induced with
1 mM IPTG as described in preceding examples l otal and soluble protein extracts were generated fi om cells removed betoie and after IPTG induction and wete resolved on a 1 2 5 % SDS-PAGE gel and stained with Coomassie blue I his analysis revealed that high levels of soluble GroEl and GroES proteins were made in the induced cells. These results demonstrated that the chaperone hyper-expression system was functional
EXAMPLE 33 Growth Of BotA/pACYCGro Cell Lines In Fermentation Cultures
Induction of BL2 KDE3) cells lacking the LysS plasmid which contained BotA expression constructs grown in shaker flask or fermentation culture resulted in the expression of primarily insoluble BotA protein. I ermentation cultures were performed to determine if the simultaneous overexpression of the Gro operon and recombinant C hoiulinum type A jirotems ( BotA proteins) resulted in enhanced solubility of the recombinant BotA protein I his example involved the fermentation of pHιsBotA(sy n)kan laclq T7]ac/pACYCGro BE2 K DE3 ) and pHtsBotA( syn )kan laclq T7/pACYC(ϊro BE2 K DE3) cell lines I he lermentations w ei e repeated exactly as described in Example 3 1 Chloramphenicol (34 μg/ml ) w as included in the feeder and fermentation cultures
a) Fermentation Of pHisBotA(syn)kan laclq TTIac/pACYCGro
BL2 I (I)E3) Cells
1 or lermentation of cells containing plasmids comprising the T71ac promoter. induction was w ith 2 gms IP FG at 1 hr post initiation of glucose feed. I he OD,,,,, was 35 at lime ol induction, then 48.5. 61 5. 67 at 1 -3 hrs post induction Viable colony counts decreased ti om 0-3 hr induction [21 ( 1 3 ). 0. 0. 0. dilution 3 utilized 3 μl of dilution 2 cells] w ith numbei s in parenthesis for the indicating microcolonies Ol 28 colonies scored at the time ol induction. 23 retained the pl lιsBotA(syn)kan laclq T71ac plasmid (kan resistant ). 22 contained the chaperone plasmid (chloramphenicol resistant) and no colonies at induction gi ew on I PTG-t an plates ( no mutations detected). These lesults were indicative of very strong promoter induction, since colony viability dropped immediately after induction l otal and soluble extracts were resolved on a 12 5% SDS-PAGE gel and stained with Coomassie High lev el induction of Gro chaperones was observed, but very low level expi ession ol soluble BotA protein was observed, increasing fi om I to 4 0 hrs post induction
( no expression detected in uninduced cells). The dramatically lower expression ol the BotA antigen in the presence of chaperone may be due to promoter occlusion ( i e . the strongei F7 promote! on the chaperone plasmid is preferentially utilized ) b) Fermentation Of pHisBotA(syn)kan laclq T7/ pACYCGro
BL21(DE3) Cells
A fermentation utilizing the T7-dπven BotA expression piasmid was performed Induction was with 1 gm IPTG at 2 hrs post initiation ot glucose Iced The ODh00 was 41 at time ot induction, then 51 5. 61 5. 61 5 and 66 at 1 -4 hrs post induction Viable colony counts decreased from 0-4 hrs induction [71. 1 (34). 1 ( 1 ). 1 . 0. dilution 3 utilized 6 μl dilution 2 cells) with numbers in parenthesis for the uninduced timepoint indicating nuciocolonies Ol 65 colonies scored at the time oi induction, all 65 retained both the pHιsBotA( syn)kan laclq J_7 plasmid (kan resistant) and the chaperone plasmid (chloramphenicol resistant) and no colonies at induction grew on IPTG+Kan plates (no mutations detected) l tal and soluble extracts weie resolved on a 12 5% SDS-PΛGL gel and stained w ith Coomassie High level induction ot Gro chaperones and moderate level expression of soluble BotA protem was observ ed, increasing trom 1 to 4 0 hrs post induction ( no expression detected in uninduced cells)
\ PLI-claπfled lysate (0 2% final cocnentration PFI ) [850 ml irom MO gm cell pellet ( 2 liter s fermentation harvest)] was purified on a large scale IF)Λ column total ol 78 mg ot protein vv as eluted I xtracts trom the purification were resolv ed on a 1 2 5% SDS-PAGF gel and stained with C oomassie The elution was found to contain an approximately 1 I mix ot BotA chaperone piotein ( Tigure 32) PEI lysates prepared in this manner were tv pically 16
OD,Sι,/ml This was estimated to be 8 mg protciivml of ly sate ( bv B A assav ) Fhus. the eiuted recombinant BotA protein tepresented 0 55% ol the total soluble cellular piotein applied to the column
In F igure 32. lane 1 contains molecular weight mai keis. lanes 2-9 contain extracts f i m pl lιsBotA( syn )kan laclq T 7/pACYC'Gro/BI 21 (DF 3) cells betore oi during purification on the I DA column Eane 2 contains total protein extract, lane 3 contains soluble piotein extract, lanes 4 and 5 contain PFl-claπfied lysates (duplicates), lanes 6 and 7 contain flow- through li om the IDA column (duplicates) and lanes 8 and 9 contain IDA column elute ( lane 9 contains U1 0 the amount applied to lane 8) I hese l esults demonstrate, that although the majority ot ihe BotA protein produced was insoluble. 20 mg/litei ol soluble iecombinant BotA protein can be purified utilizing the pHιsBotA( sy n)kan laclq T7/pACYCGro/BL21 (DL3 ) expression sv stem EXAMPLE 34
Purification Ot Recombinant BotA Protein F rom Folding Chaperones
In this example of size exclusion chromatography was used to puπfv the recombinant BotA piotein avvav from the folding chaperones and imidazoie present in the IDA-purified male l ( f x 33 ) l o enhance the solubility of the i ecombinant BotA protein during scale-up. the piotein was co-expressed with folding chaperones (Ex 33) Λs observed with the recombinant BotB protein ( I xample 40 below ), the folding chaperones co-eluted with the iecombinant BotA protem during the Ni-IDA purification step Because the recombinant BotA and BotB pioteins hav e similar molecular weights (about 1 /10 the size ot the non-reduced folding chapeione) and the imidazoie step gradient strategy was unsuccessful in purifying BotB away Irom the f olding chaperone ( see I x 40). size exclusion chromatography vvas examined tor the abilitv to puπ l v the recombinant BotA protein awav from the tolding chaperones \ column (2 5 x 24 cm ) containing Sephacry l S- 100 HR (Pharmacia) was poured (bed v olume 1 10 ml ) Proteins hav ing molecular weights gieater than 100 K ai e expected to elute in the v id v olume under these conditions and smaller proteins should be retained by the beads and elute at dif ferent times, depending on their molecular weights l maintain solubilitv ot the purified BotA protem, the Sephacry l column was equilibrated in a buffer having the same salt concentration as the buffer used to elute the BotA protein trom the IDA column ( / e . 50 mM sodium phosphate. 0 5 M NaCl. 10% glycerol. all reagents tiom Mallinki odt. hesterfield. MO)
1 ιv e nulliliters ot the I DA-purified recombinant BotA protein ( Ex 33 ) was filtered through a 0 45 μ syringe filter, applied to ihe column and the equilibration buffer was pumped thi ough the column at a flow rate of 1 ml/minute Eluted proteins were monitored by absorbance at 280 nm and collected either manually or with a fraction collector (BioRad) Appropriate tractions were pooled, if necessary, and the protein vvas quantitated bv absorbance at 280 nm and/or BCA protein assay ( Pierce) T he isolated peaks weie then analy zed bv native and/or SDS-PAGE to identitv the proteins present and to ev aluate purity I he f olding chaperone eluted first, followed by the recombinant BotA protein and then the imidazoie
SDS-PAGE analysis ( 12 5% polyacrylamide. reduced samples) was used to ev aluate the punty ot the IDA-purified iecombinant BotA protein before and after S- 100 purification I igure 33 shows the difference in purity before and alter the S- 100 purification step In Figure 33 lane 1 contains molecular weight markers (BioRad broad range) Lane 2 shows the IDA-purtfied recombinant BotA protein preparation, which is contaminated with significant amounts of the tolding chaperone 1 ollowing S- 1 00 purification, the amount ol folding chapeione present in the BotA sample is reduced dramatically (lane 3) Lane 4 contains no piotein (/ e . it is a blank lane), lanes 5-8 contain samples ot IDΛ-puπfied lecombmant BotB and BotL proteins and are discussed infia
1 ndotoxin levels in the S- 100 purified BotA prepaiation were determined using the LAI assav (Associates ol Cape Cod) as describe in Example 24 I he purified BotA preparation was found to contain 22 7 to 45 5 EU/mg recombinant protem
Fhese results demonstiate that size exclusion chi omatography was successful in puniv ing the iecombinant BotA protein from folding chaperones and imidazoie following an initial I DA purification step f urthermore, these results demonstrate that the S- 100 purified BotA pi otein was substantially f iee of endotoxin
EXAMPLE 35
C loning And Expression Ot T he C Fragment Of The hoiulinum Seroty pe B l xin Gene
I he ( botulinum ty pe B neurotoxin gene has been cloned and sequenced [ Whelan et al ( 1992) Appl I nviron Microbiol 58 2345 and Hutson et al ( 1994) C ui r Miciobiol 28 101 ] I he nucleotide sequence ot the toxin gene deriv ed liom the I klund 1 7B strain ( λ TC C 25765 ) is available liom the L MBL/GenBank sequence data banks under the accession number X71343. the nucleotide sequence ot the coding region is listed in SI Q ID NO 39 I he ammo acid sequence of the C botulinum type B neurotoxin deiived trom the strain I klund 17B is listed in SLQ ID NO 40 I he nucleotide sequence of the ( " hoiulinum seroty e B toxin gene derived trom the Danish strain is listed in SI Q ID NO 41 and the corresponding ammo acid sequence is listed in SEQ ID NO 42
I he DNA sequence encoding the native ( hoiulinum serotv pe B ( Iiagment gene deriv ed fi om the Eklund 1 7B strain can be expressed using the pET l lisb v ectot . the resulting coding region is listed in SI Q I D NO 43 and the coi respondmg ammo acid sequence is listed in SEQ ID NO 44 The DNA sequence encoding the native hoiulinum seroty pe B C Ii agment gene dei ived tiom the Danish sttain can be expressed using the pETHisb vector, the lesulting coding region is listed in SEQ ID NO:45 and the corresponding amino acid sequence is listed in SEQ ID NO:46. The C frgament region from any strain of C. hoiulinum serotype B can be amplified and expressed using the approach illustrated below using the C fragment derived from C. hoiulinum type B 201 7 strain. The ( '. hoiulinum type B neurotoxin gene is synthesized as a single polypeptide chain which is processed to form a dimer composed of a light and a heavy chain linked via disulfide bonds: the type B neurotoxin has been reported to exist as a mixture of predominallv single chain with some double chain ( Whelan et al.. supra). The 50 kD carboxy-tcrminal portion of the heavy chain is referred to as the C fragment or the Flt- domain. Expression of the C fragment of C. hoiulinum type B toxin in heterologous hosts (e.g.. E. coli) has not been previously reported.
The native C fragment of the (.'. hoiulinum serotype B toxin gene vvas cloned and expression constructs were made to facilitate protein expression in E. coli. This e.xample involv ed PCR amplification of the gene, cloning, and construction of expression vectors. I he C fragment of the C botulinum serotype B ( BotB) toxin gene was cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. Fhe ( ' hoiulinum ty pe B 2017 strain was obtained from the American Ty pe Culture Collection ( ATCC ~ I 7843). The following primer pair was used to amplify the BotB gene: 5"-C'GCCΛTGGCTGATACAΛTACTAATAGAA ATG-3' [5" primer, engineered Neol site underlined ( SEQ ID NO:47)[ and 5'-GCAAG C T7TΛTTCAGTCCΛCCCTTCΛTC-3 " [ 3' primer, engineered /-//mil 11 site underlined, native gene termination codon italicized (SEQ ID NO:48 ) j. After cloning into the pCRscript vector, the Λ77tT(tllled )/// dl I I fragment vvas cloned into pETHisb vector as described for BotA C fragment gene in Example 28. The resulting construct vvas termed pHisBotB. pHisBotB expresses the BotB gene sequences under the transcriptional control of the
T7 lac promoter and the resulting protein contains an N-terminal l OXHis-tag affinity tag. The pHisBotB expression construct vvas transformed into BL21 (DE3) pLysS competent cells and 1 liter cultures w ere grown, induced and his-tagged proteins were purified utilizing a NiNTA resin (eluted in low pi I elution buffer) as described in Example 28. T otal, soluble and purified proteins were resolved by SDS-PAGE and detected by Coomassie staining and
Western blot hybridization utilizing a chicken anti-C. hoiulinum serotype B toxoid primary- antibody (generated by immunization of hens using ( '. hoiulinum seroty pe B toxoid as described in Example 3). Samples of BotA and BotE C fragment proteins w ere included on
: 89 the gels for MW and immunogenicity comparisons Strong immunoreactivity to only the BotB piotein was detected with the antι-( botulinum serotvpe B toxoid antibodies The recombinant BotB protein was expressed at low levels (3 mg/hter) as a soluble protem Fhe purified BotB piotem migrated as a single band ot the predicted MW (/ e 5()kD) Ihese iesults demonstrate the cloning of the native ( hoiulinum serotvpe B C
Iiagment gene the expression and purification of the recombinant BotB protein as a soluble his-tagged protein in E coli
EXAMPLE 36 Generation Of Neutralizing Antibodies Using The Recombinant pFlisBotB Protein
The abilitv of the purified pHisBot protein to generate neutralizing antibodies was examined Nine BAI Be mice were immunized with BotB piotein (purified as described in 1 \ 3 ) using Geibu GMDP adjuvant (CC Biotech) Ihe low pll elution was mixed with Gerbu adjuvant and used to immunize mice Each mouse received a subcutaneous injection ol 100 μl antιgen/ad|uvant mix (15 μg antigen + 1 μg adjuvant) on dav 0 Mice were subcutaneoLislv boosted as above on da\ 14 and bled on dav 28 Mice weie subsequentlv boosted 1-2 weeks altei bleeding and weie then bled on dav 70
Anti-C hoiulinum seiotype B toxoid titers were determined in dav 28 serum trom individual mice irom each gioup using the TLISA protocol outlined in 1 xample 29 with the exception that the plates were coated with ( hoiulinum seiotvpe B toxoid and the pπmarv antibodv vvas a chicken anti-C hoiulinum serotvpe B toxoid Seioconversion [relative to control mice immunized with pHisBot! antigen (desciibed below)) was observed with all 9 mice immunized with the punfied pHisBotB protein Ihe abilitv ot the anti-BotB antibodies to neutralize native C hoiulinum tvpe B toxin vvas tested in a mouse-C hoiulinum neutralization model using pooled mouse serum (see 1 x 23b) Ihe I D,, oi purified ( hoiulinum type B toxin complex (Di Lπc lohnson Universitv of Wisconsin Madison) was determined bv a intraperitoneal (IP) method [Schantz and kautler (1978) supia] using 18-22 g lemale ICR mice The amount ol neutralizing antibodies present in the seium ot the immunized mice was determined using seium antibodv titrations Ihe various seium dilutions (001 l) were mixed with 5 LD,„ units ol ( hoiulinum tvpe B toxin and the mixtures were injected IP into mice Ihe neutralizations were performed in duplicate Ihe mice were then obseived toi si-ins of botulism foi 4 davs Undiluted serum (dav 28 or day 70) was found to protect 100% of the injected mice while the 1 : 10 diluted serum did not. This corresponds to a neutralization titer of 0.05-0 5 lU/ml
These results demonstrate that seroconversion occurred and neutralizing antibodies were induced when ihe pHisBotB protein was utilized as the immunogen
EXAMPLE 37
Construction Of Vectors To Facilitate Expression Ot Ilis-Tagged BotB Protein In Fermentation Cultures
Λ number of expression v ectors were constructed to facilitate the expression of i ecombinant BotB protein in large scale fermentation culture. T hese constructs varied as to the stiength ot the promoter utilized ( 7 or T7lac) and the presence of repressor elements ( laclq ) on the plasmid I he resulting constructs varied in the level oi expression achieved and in plasmid stability w hich facilitated the selection ot a optimal expression sy stem for iermeniation scaleup
I he BotB expression v ectors created for fermentation culture were engineered to utilize the kanamvcin rather than the ampiciilin resistance gene, and contained either the T7 or F7lac promoter , w ith or w ithout the laclq gene for the reasons outlined in Example 30
I n all cases, the protein expressed by the various expression vectors is the pHisBot B protein desci ibed in Example 35. w ith the only differences between clones being the alteration ot v t ious regulatory elements Using the designations outlined below, the pHisBotB clone ( 1 x 3 ) is equiv alent lo pHisBotB amp F7lac
a) Construction Of pHisBotB kan T7lac pHisBotB kan T71ac was constructed by insertion of the Bg/l /Hiπdlll fragment ot pHisBotB which contains the BotB gene sequences into the pPA l 870-2680 kan T 71ac vector which had been digested with Bglll and Hindlll (the pPA 1870-2680 kan I 7lac vector contains the pET24 kan gene in the pET23 vector, such that no laclq gene is present) Proper construction ot pHisBotB kan T71ac was confirmed by restriction digestion b) Construction Of pHisBotB kan laclq T7Iac pHisBotB kan laclq T7Iac was constructed by insertion ot the Bglll'Hindlll fragment ol pHisBotB which contains the BotB gene sequences into similar l\ cut pFT24a vector Proper construction of pHisBotB kan laclq 171ac was con fumed by restriction digestion
c) Construction Of pHisBotB kan laclq T7 pHisBotB kan laclq T7 was constructed by inserting the Ndel/Xhol Iiagment fiom plIisBotT kan laclq 171ac which contains the BotB gene sequences into similarlv cleaved pPA 1870-2680 kan laclq T7 vector (this vector contains the T7 piomoter. the same N- terminal his-tag as the Bot constructs, the ( difficile toxin A inseit and the kan laclq genes, this cloning replaces the ( difficile toxin A insert with the BotB insert) Proper construction vvas confirmed by restriction digestion
I xpression of recombinant BotB protein trom these expression vectors and purification ot the BotB piotein is described in Fxample 38 below
EXAMPLE 38
I ermentation And Puiification Ot Recombinant BotB Piotem Utilizing Ihe pHisBotB kan laclq T71ac. pHisBotB kan I 71ac And pHisBotB kan laclq 17 Vectors
Fhe pFlisBotB kan laclq T7lac. pHisBotB kan I 71ac and BotB kan laclq T7 constructs
[all lianstormed into the B12KD13) stiain] were grown in fermentation cultures to determine the utility ol the various constructs lor laige scale expression and puiification ol soluble BotB protein \11 leimentations were peitormed as described Fxample 31
a) Fermentation Of pHisBotB kan laclq T71ac/B121(DE3) Cells
The termentation cultuie was induced 45 m post start ot glucose iced with 1 gm IPTG (final concentration = 04 mM) pH vvas maintained at 65 rather than 70 Ihe OD,,(1(, was 27 at lime ot induction, then 35.38. and 40 at 1-3 hrs post induction Duplicate platings ot diluted 1 hr induction samples (dilutions were prepared as described I x 31 dilution 3 utilized 3 μl ot dilution 2 cells) on ISA and L B t-kan plates vieided 89 1 SΛ colonies and 81 kan colonies (90% kan resistant) lotal and soluble protein extracts were resolved on a 125% SDS-PAGE gel and total protein vvas detected by staining with oomassie blue I ow level induction ot msoluble Bot B protein vvas observed, increasing from 1 to 3 hrs post induction (no expression was detected in uninduced cells).
b) Fermentation Of pHisBotB kan T7lac/BI21(DE3) Cells Fhe fermentation culture was induced I hr post start of glucose feed with 2 gm IPTG
( final concentration = 0.8 mM). pH was maintained at 6.5 rather than 7.0. Fhe OD,,(H, was 24.5 at time of induction, then 31 .5. 32. and 33 at 1 -3 hrs post induction, respectively. Duplicate platings of diluted 0 hr and 2 hr induction samples (dilutions were prepared as described Ex. 3 1 : dilution 3 utilized 3 μl of dilution 2 cells) on TSA and LB+kan plates y ielded 32 TSA colonies and 54 kan colonies (all kan resistant) for uninduced cells, and 1
T SA colon and 0 kan colonies 2 hr post induction. T hese results were indicative of strong induction, since v Table counts decreased dramatically 2 hrs post induction. otal and soluble extracts were resolved on a 10% SDS-PAGE gel and total protein w as detected by staining w ith Coomassie blue. Moderate induction of insoluble BotB protein was observed, increasing from I to 3 hrs post induction (no expression was detected in uninduced cells).
c) Fermentation Of pHisBotB kan laclq T7/B121(DE3) Cells
The fermentation w as induced 2 hr post start of glucose feed with 4 gm I PTG ( final concentration = 1 .6 mM ). pH was maintained at 6.5 rather than 7.0. T he OD,„,(, was 45 at time of induction, then 47. 50. and 50 and 55 at 1 -4 hrs post induction, respectively. Viable colon counts decreased after induction (96. 1. 1. 2. 3: dilution 3 utilized 3 μl of dilution 2 cells ). Of 63 colonies scored at the time of induction, al! 63 retaining the BotB plasmid (kan resistant) and no colonies at induction grew on IPTG + Kan plates ( no mutations detected). Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and total protein was detected by staining with Coomassie blue. Moderate level induction of insoluble BotB protein w as observed, increasing from 1 to 4 hrs post induction (lower level expression was detected in uninduced cells, since the T7 rather than T7lac promoter was utilized).
d) Purification Of pHisBotB Protein From pHisBotB amp
T7lac/B121(DE3) Cells
Soluble recombinant BotB protein was purified utilizing NiNTΛ resin from 80 ml of cell lysate generated from cells harvested from a pHisBotB fermentation [using the pHisBotB amp T71ac/B121 (DE3 ) strain]. As predicted from the smail scale results above, the majority of the induced protein was insoluble. As well, the eluted material was contaminated with multiple E. coli contaminant proteins. A Coomassie blue-stained SDS-PAGE gel containing extracts derived from pHisBotB amp T71ac/B121 (DE3) cells before and during purification is shown in Figure 34. In Figure 34. lane 1 contains broad range protein MW markers
( BioRad). Lanes 2-5 contain extracts prepared from pHisBotB amp T71ac/B121 (DE3 ) ceils grown in fermentation culture: lane 2 contains total protein: lane 3 contains soluble protein: lane 4 contains protein which did not bind to the NiNTA column (i. e.. the flow-through ) and lane 5 contains protein eluted from the NiNTA column. Similar results were obtained using a small scale IDA column utilizing a cell lysate from the pHisBotB kan laclq T7 fermentation described above. 250 mis of a 20% w/v PEI clarified Ivsate ( 50 gms cell pellet) of botB kan laclq T7/B12 KDE3 ) cells were purified on a small scale I DA column. [Tie total yield of eluted protein was 2 1 mg protein ( assuming 1 mg/ml solution : 2 OD,s„/ml). When analyzed by SDS-PAGE and Coomassie staining, the BotB protein was found to comprise approximately 50% of the eluted protein w ith the remainder being a ladder of £. coli proteins similar to that observ ed with the NiNT A purification.
Fhe NiNT A alkaline phosphatase conjugate w as utilized to detect his-tagged proteins on a Western blot containing total, soluble, soluble ( PEI clarified), soluble ( after IDA column) and elution samples from the IDA column purification. The results demonstrated that a small percentage of BotB protein was soluble, that the soluble protein was not precipitated by PEI treatment and was quantitatively bound by the IDA column. Since a 1 liter fermentation harvest yielded a 67.5 gm cell pellet, this indicated that the yield of soluble affinity purified BotB protem from the I DA column as 14 mg/litcr.
EXAMPLE 39
Co-Expression Of Recombinant BotB Proteins
And Folding Chaperones I n Fermentation Cultures
Fermentations were performed to determine if the simultaneous overexpression of folding chaperones ( i. e.. the Gro operon ) and the BotB protein resulted in enhanced solubility of the Bot B protein. This example involved fermentation of the pl iisBotBkan laclq T71ac/pACYCGro BL21 ( DE3). pHisBotB kan T71ac/pACYCGro B12 KDE3) and pl iisBotBkan laclq T7/ pACYCGro BL21 (DE3) cell lines Fermentation was carried out as described in Example 31 34 μg/ml chloramphenicol was included in the feeder and fermentation cultures
a) Fermentation Of pHisBotBkan laclq T7lac/pACYCGro BL21 (DE3) Cells
Induction vvas with 4 gms IPTG at I hr 1 5 nun post initiation ot the glucose feed I he OD, „„ was 38 at time of induction then 50. 58 5. 62 and 68 at 1 -4 hrs post induction Viable colonv counts decreased during induction (24. 0 0 2 0 at 0-4 hr induction dilution 3 utilized 3 μl of dilution 2 cells) Oi 24 colonies scored at the time of induction 24 retained the BotB plasmid (kan resistant) 24 contained the chaperone plasmid (chloramphenicol l esistant ) and no colonies at induction grew on IPTCH kan plates ( no mutations detected) l otal and soluble extracts were resolved on 12 5% SDS-PAGF gels and were either stained w ith ( oomassie blue or subjected to Western blotting ( his-tagged pioteins were detected utilizing the NiN lA-alkaline phosphatase conjugate) I his analvsis revealed that the Gt o chapeioncs were induced to high levels but v erv low level expression ot soluble BotB pi otein vvas observed increasing ti om I to 4 0 his post induction ( no expression detected in uninduced cells induced protein detected onlv on Western blot) I he diamaticallv lower expiession ol BotB protem in the presence of chaperone mav be due to promoter occlusion U e the sti onger 1 7 promoter on the chaperone plasmid was preferentiallv utilized)
b) Fermentation Of pHisBotB kan T7lac/pACYCGro/BI2I(DE3) Cells
I nduction vvas with 4 gms I PTCJ at 1 hr post initiation ol the glucose teed T he ODWM) was 33 s at time ot induction then 44 5 1 . 58 5 and 69 at 1 -4 hrs post induction Viable colonv counts decreased after 2 hrs induction (43 65. 74. 0 (70) 0 (70) at 0-4 hr induction bracketed numbers represent microcolonies. dilution 3 utilized 3 μl of dilution 2 cells) Most colonies at induction retained the BotB plasmid (kan resistant )and the chaperone plasmid (chloi amphemcol l esistant) and no colonies at induction grew on IPTG+ Kan plates ( no mutations detected) l otal and soluble extracts weie resolved on a 12 5% SDS-PAGF gel and subjected to Westei n blotting, his-tagged proteins were detected utilizing the NiNTA-alkahne phosphatase conjugate This analvsis revealed that the Gro chaperones were induced to high lev els and low level expression of soluble Bot B protein was observed, increasing from i to 4.0 hrs post induction ( no expression detected in uninduced cells)
A small scale IDA purification of BotB protem from a 250 ml PFI clarified 1 5% w/v extract ( 37 5 gm cell pellet) yielded approximately 1 2 5 mg protein, ol w hich approximatelv 50% vvas BotB protein and 50% was GroEL chaperone (assessed by Coomassie staining of a
10% SDS-PAGE gel) T he NiNTA alkaline phosphatase conjugate was utilized to detect his- tagged proteins on a Western blot containing total, soluble, soluble (PEI clarified), soluble ( aftei I DA column) and elution samples trom the IDA column purification The results demonstrated that all of the BotB protein produced by the pHisBotB kan T71ac/pΛCYCGio/BI21 (DE3) cells was soluble, the BotB protein vvas not precipitated by PEI ti eatment and was quantitatively bound by the IDA column Since a 1 liter fermentation harvest y ielded a 75 gm cell pellet, this indicated that the ield ol soluble alfinity purified bot 13 piote liom this fermentation was 12.5 mg/hter I hese results also demonstrated that additional purification steps are necessary to separate the chaperone proteins trom the BotB protein
c) Fermentation Of pHisBotBkan laclq
T7/pAC YCGro/BL21 (DE3) Cells
I nduction vvas with 4 gms IPTG at 2 hr post initiation ol the glucose teed I he C)D,,„0 was 46 at time of induction, then 56. 63. 69 and 71 5 at 1 -4 hrs post induction Viable colony counts decreased after induction ( 58. 3( 5). 3. 0. 0 at 0-4 hr induction, bracketed numbers l epresent micr ocolonies. dilution 3 utilized 3 μl of dilution 2 cells ) All (53/53 ) colonies scored at the time of induction retained the BotB plasmid ( kan resistant) and the chaperone plasmid ( chloramphenicol resistant ) and no colonies at induction grew on IP I G+kan plates ( no mutations detected)
Total and soluble extracts were resolved on a 10% SDS-PAGF. gels and Western blotted and his-tagged proteins were detected utilizing the NiN I A-alkaline phosphatase conjugate This analysis revealed that the Gro chaperones were induced to high levels (observ ed by ponceau S staining), and a much higher expression ol soluble Bot B protem ( compared to expression in the pHisBotB kan T71ac/pΛCYCGro fermentation) was observed at all timepoints. including uninduced cells (some increase m BotB protein levels w eie observed after induction) A small scale IDA purification of BotB protein from a 100 ml PEI clarified 15% w/'v extract ( 15 gm cell pellet) yielded approximately 40 mg protein, of which approximately 50% was BotB protein and 50% was GroEL chaperone. as assessed by Coomassie staining of a 10% SDS-PAGE gel. The NiNTA alkaline phosphatase conjugate was utilized to detect his- tagged proteins on a Western blot containing total, soluble, soluble (PEI clarified), soluble
( after I DA column) and elution samples from the IDA column purification. The results demonstrated that a significant percentage (i. e.. - 10-20 %) of BotB prolein vvas soluble, that the solubiiized protein was not precipitated by PEI treatment and was quantitatively bound by the IDA column. Since a 10 liter fermentation yielded a 108 gm cell pellet, this indicated that the yield of soluble affinity purified BotB protein from this fermentation was 144 mg/liter.
In a scale up experiment. 2 liters of a 20% w/v PEI clarified lysate of pHisBotB kan laclq 1 7/pACYCGro/BL21 (DE3) cells were purified on a large scale IDA column. The puri fication was performed in duplicate. The total yield of BotB protein vvas 220 and 325 mgs protein in the two experiments (assuming I mg/ml solution - 2.0 OD:s /ml ). This represents 0.7% or 1 .0%. respectively, of the total soluble cellular protein (assuming a PEI ly state having a concentration of 8 mg protein/ml and that the eluted material comprises a 1 : 1 mixture of BotB and folding chaperone). T he NiNTA alkaline phosphatase conjugate vvas utilized to detect his-tagged proteins on a Western blot containing total, soluble, soluble (PEI clarified ), soluble ( after IDA column ) and elution samples from the IDA column purification. These results demonstrated that a significant percentage (i. e.. - 1 0-20 % ) of the BotB protein was soluble, that the solubihzed protein vvas not precipitated by PEI treatment and was quantitativ ely bound by the IDA column. Since a 1 liter fermentation harvest yielded a 108 gin cell pel let, this indicated that the yield of soluble affinity purified BotB protein from the large scale purification was 60 mg or 89 mg/liter. These results also demonstrated that further purification would be necessary to remove the contaminating chaperone protein.
The above results provide methodologies for the purification of soluble BotB protein from fermentation cultures, in a form contaminated predominantly with a single E. coli protein ( the folding chaperone utilized to enhance solubility). In the next example, methods are provided for the removal of the contaminating chaperone protein. EXAMPLE 40
Removal Of Contaminating Folding Chaperone Protem From Purified Recombinant C hoiulinum I y pe B Protein
5 In this example size exclusion chromatography and ultrallltration was used to puπfv iecombinant BotB protein from the folding chaperones and imidazoie in IDA-purified mater ial l enhance the solubility of the recombinant BotB protein during scale-up. the protein was co-expressed with folding chaperones (see Ex 39) During the Ni-IDA pui ification step. 10 the folding chaperones co-eluted with the BotB protein in 800 mM imidazoie. therefore, a second pui ification step was required to isolate the BotB f ree of folding chapeiones I ane 3 of Figure 35 contains proteins eluted from an IDA column to w hich a lysate ot pHisBotB kan laclq I 7/pΛCYCGro/BL21 (DE3) cells had been applied, the proteins were lesolved on a 4- 15% pol aci y lamrde pre-cast gradient gel ( Bio-Rad. Hercules. C Λ ) run under nativ e I s conditions and then stained with Coomassie blue In I igure 35. lanes I and 4 contain proteins piesent in peak 1 and peak 2 f rom a Sephacryl S- 1 00 column run as desci ibed below , lane 2 is blank
As seen in lane 3 of Figure 35. the IDA-purified sample consists pπmaπlv of the folding chaperones and the BotB protein I he lact that the chaperones and the Bot B antigen 0 appear as tw o distinct bands under native conditions suggested they were not complexed togethei and therefore, it should be possible to separate them, using either a giadient ol imidazoie concentrations oi size exclusion methods
I n order to determine whether a gradient ol imidazoie concentrations could be used to separate the chaperone liom the BotB protein, a step gradient using imidazoie at 200. 400. 25 600. and 800 mM in 50 mM sodium phosphate. 0 5 M NaG and 10 % glycerol. pll 6 8 was applied to an IDA column (containing proteins bound from a lysate of pHisBotB kan laclq r7/pACYCGιo/BL21 (DL 3 ) cells) Bv narrowing the range ol imidazoie concentrations, it was hoped that the BotB and chaperone proteins would diffeientiallv elute at different concentrations ot imidazoie Eluted proteins were monitoi ed bv absorbance at 280 nm and 30 collected eithei manuallv or with a fi action collector ( BioRad) Piote was iound to elute at
200 and 400 mM imidazoie only
I igure 36 shows a Coomassie stained SDS-PAGE gel containing protem eluted during the imidazoie step giadient 1 ans 1 contains broad range MW markei s ( BioRad) Lane 2 contains BotB protein purified by IDA chromatography of an extract of pHιsBotB/BL21 (DE3) pLysS cells grown in shaker flask culture (/ e . no co-expression of chaperones. Ex 35) Lane 3 contains a 20% w/v PEI clarified lysate of pHisBotB kan laclq T7/pAC YCGro/BL21 (DE3) ceils (/ e . the lysate prior to purification by IDA ^ chiomatograph ) Lanes 4 and 5 contain protein which eluted at 200 oi 400 mM imidazoie, icspectiv elv Lane 6 is blank Lanes 7 and 8 contain 1 /5 the load present in lanes 4 and 5 \s show n in Tigure 36. both the chaperone and the BotB protein eluted in 200 mM imidazoie. and more chaperone elutes in 400 mM imidazoie. however no concentration ot imidazoie tested pei mittcd the elution ot BotB protein alone C onsequently , no significant
10 pui ification was achieved using imidazoie at these concentrations
Because of the considerable dilfeience in molecular weights between the folding chapeione which is a multimei w ith a total molecular weight around 400 kD (as determined on a Shodex KB 804 sizing column bv HPLC). and the recombinant BotB pi otein (molecular weight ai ound 5() kD). size exclusion chromatographv was next examined tor the ability to
I 5 sepaiate these proteins
a) Size E clusion hromatograph}
\ column containing Sephacrv I S- 100 HR (S- 100) (Pharmacia) was poui ed (2 5 cm x 24 cm 1 1 0 ml bed volume) The column was equilibrated in a buf fei consisting of 0 phosphate bulfered saline ( l OinM potassium phosphate. 150 mM NaCl pi I 7 2) and 10 % gly cei ol ( Mallinkrodt) U picallv 5 ml of the IDA-purified BotB protein was filtered through a 0 45 μ sv πnge filter and applied to the column, and the equihbiation butler was pumped thiough the column at a flow rate of 1 ml/minute Eluted proteins were monitored bv absoi bance at 280 nm and collected eithei manually oi with a ii action collector Appropπate
2s tubes w ere pooled, if necessary , and the protein was quantitated by absorbance at 280 nm and/or by BCA protem assay I he isolated peaks were then analy zed by native and/or SDS-PAGE to identify the protein and ev aluate the purity
Because ot its larger size, the tolding chaperone eluted fu st lollowed bv the i ecombinant BotB protein A smallei thud peak was obsei ved which failed to stain when
30 analy zed by SDS-PAGE. and thereloie vvas presumed to be imidazoie
SDS-PAGE analysis ( 12 5% poly aery lamide. leduced samples) was used to ev aluate the purity of the IDA-punfied recombinant BotB protein before and aftci S- 100 purification Fhe lesuits aie shown in Figure 33 In Figure 33. lane 1 contains broad range MW markers ( BioRad) Lane 5 contains IDA-purified BotB protein Lane 6 contains IDA-purified BotB protein following S- 100 purification. Lane 7 is blank ( lanes 2-4 were discussed in Ex 34 above)
T he results shown in Figure 33 show that the IDA-pui ified BotB is significantly contaminated with the folding chaperone ( molecular weight about 60 kD under reducing conditions, lane 6) Following S- 100 purification, the amount ol folding chaperone present in the BotB sample was reduced dramatically ( lane 7) Visual inspection of the Coomassie stained SDS-PAGE gel revealed that after S- 100 purification. - 90% of the total protein present was BotB. The IDA-purified BotB and the S- 100-puπfied BotB samples were analyzed by HPLC on a size exclusion column ( Shodex KB 804); this analysis revealed that the BotB protem represented 64% of the total protein in the IDA-purified sample and that follow ing S- 100 purification, the BotB protein represented '95% of the total protein in the sample
The I DA-purified BotB material w as also applied to a ACA 44 ( SpectraPor. Houston. I X) column T he ACA 44 resin is equivalent to the S- 100 resin and chromatography using the ΛC'A 44 resin was carried out exactly as described above loi the S- 100 resm I he ACA 44 resin was found to separate the recombinant BotB protein trom the folding chaperone I he ΛC'A 44-puπfied BotB sample was analyzed for endotoxin using the L ΛI assay (Associates of Cape Cod) as describe in Example 24 I wo ahqouts ot the ΛC Λ 44-puπfιed BotB preparation were analyzed and were Iound to contain either 3 to 1 16 1 U/mg recombinant protein or 94 to 1 9 FU/mg recombinant protein
1 hese results demonstrate that size exclusion chromatogiaphv can be used to purify the i ecombinant BotB protem li om the lolding chaperone and imidazoie in I D puπfied material
b) Ultrafiltration For The Separation Of Recombinant BotB
Protein And Chaperones
Ultrafiltration was examined as an alternative method lor the separation recombinant BotB protein and folding chaperones in IDA-purified material While in this example only mixtures ol BotB and chaperones were separated by ultrafiltiation. this technique is suitable lor use w ith recombinant BotA and BotE. proteins as well piovided that the w ash bultei s used are altered as necessary to take into account dif ferent requirements toi solubility
Fhe recombinant BotB protein and folding chaperones were separated using a two-step sequential ultrafiltration method. Fhe first membrane used had a nominal molecular weight cutoff (MWCO) of approximately 100 kD; this membrane retains the larger folding chaperone while allowing the smaller recombinant protein to pass through. The addition of several volumes of wash buffer may be required to efficiently wash the recombinant protein through the membrane. T he second step utilized a membrane with a nominal MWCO of approximately 10 kD. During this step, the recombinant antigen was retained by the membrane and could be concentrated to the degree desired and the imidazoie and excess wash buffer passed through the membrane.
Twenty-seven inilliliters of an IDA-purified BotB preparation was ultrafiitered through a 47 mm YM 100 ( 100 kD MWCO) membrane (Amicon) in a 50 ml stirred cell (Amicon). Fhe membrane vvas washed in dd I LO prior to use as recommended by the manufacturer. Six volumes of 10% glycerol in PBS were washed through to remove most of the recombinant BotB protein and this wash vvas collected in a separate vessel. The resulting BotB protein-rich filtrate was then concentrated 12-fold using a YM 10 ( 10 kD MWCO) membrane ( Amicon). to a final volume of 14 ml. The YM 100 and YM 10 concentrates were analyzed along w ith the Iv sate starting material by native PAGE using a 4 - 1 5% pre-cast gradient gel
( BioRad ). I he results arc shown in Figure 37. in Figure 37. lane 1 contains IDA-purified BotB derived from a shaker flask culture (i.e.. no co-expression of chaperones: Ex. 35): lane 2 contains a 20% w/'v PEI clarified lysate of pHisBotB kan laclq T7/pΛCYCGro/BL21 (DE3 ) cells; lane 3 shows the lysate of lane 3 after I DA purification: lane 4 contains the YM 10 concentrate and lane 5 contains the YM
1 0 concentrate.
Fhe results shown in F igure 37 demonstrate tliat the recombinant BotB protein can be purified away from the folding chaperone by ultrafiltration through a 100 kD MWCO membrane ( lane 4). leaving the chaperone protein in the 100 kD concentrate ( lane 5 ). Analysis oϊ the sample in lane 5 also showed that very little of the BotB protein was retained by the 100 kD MWCO membrane after 6 volumes of wash buffer had been applied.
T he BotB samples following IDA chromatography and following ultrafiltration through the YM 1 00 membrane were anlyzed by HPLC on a size exclusion column ( Shodex KB 804): this analv sis revealed that the BotB protein represented 64% of the total protein in the IDA- purified sample and that following ultrafiltration through the YM 100 membrane, the BotB protein represented >96% of the total protein in the sample.
The BotB protein purified by ultrafiltration through the YM 100 membrane was examined for endotoxin using the LΛL assay (Associates of Cape Cod) as describe in Example 24 Two aliqouts of the YM 100-puπfied BotB preparation were analvzed and were found to contain either 18 to 36 EU/mg recombinant protein or 125 to 250 FU/mg iecombinant protein
The above results demonstrate that size exclusion chromatography and ultrafiltration can be used to punt} recombinant botuhnal toxin proteins awa} fiom folding chaperones
EXAMPLE 41
Cloning And Expression Of The C Fragment Of The C hoiulinum Serotype F I oxin Gene
Ihe C hoiulinum type F neurotoxin gene has been cloned and sequenced from several different strains [Poulet ei al ( 1992) Biochem Bιoph}s Res C ommun 183107 (strain Beluga) W helan el al (1992) Lur .1 Biochem 204657 (strain NCTC 11219).1 ujn el al (1990) Microbiol Immunol 341041 (partial sequence ot strains Mashike. Iwani and Otaru) and Eu|iι et al (1993). I Gen Microbiol 1 979 (strain Mashike)) The nucleotide sequence ot the tvpe 1 toxin gene is available liom the EMBL sequence data bank undei accession numbers X62089 (stiain Beluga) and X62683 (strain NCTC 11219) Ihe nucleotide sequence ot the coding region (stiain Beluga) is iisted in SEQ ID NO 49 Ihe ammo acid sequence of the ( hoiulinum t}j * 1 neurotoxin derived from strain Belgua is listed in SEQ ID NO 50 Ihe nucleotide sequence of the coding region (strain NCTC 11219) is listed in SEQ ID
NO 51 Ihe amino acid sequence of the ( hoiulinum tvpe 1 neurotoxin derived from strain NCTC I 1219 is Iisted in SEQ ID NO 52
Ihe DNA sequence encoding the native ( hoiulinum serotvpe F C Iiagment gene derived tiom the Beluga stiain can be expressed as a histidine-tagged protein using the pi THisb vector: the resulting coding region is listed in SFQ ID NO 53 and the corresponding ammo acid sequence is listed in SFQ ID NO 54 Ihe DNA sequence encoding the C Iiagment ot the native ( hoiulinum serotype L gene derived trom the NCTC 1121 strain can be expressed as a histidine-tagged fusion protem using the pE I llisb vector, the lesulting coding region is Iisted in SFQ ID NO 55 and the corresponding amino acid sequence is listed in SEQ ID NO 56 Ihe C fiagment region from anv stiain of C hoiulinum seiotvpe L can be amplified and expressed using the approach illustrated below using the C Iragment derived liom ( hoiulinum type E 22 1 strain (AICC #17786) The type E neurotoxin gene is svnthesized as a single polypeptide chain which may be convened to a double-chain form (it a heavy chain and a light chain) bv cleavage with trvpsin unlike the type A neurotoxin. the type E neurotoxin exists essentially onlv in the single-chain form Ihe 50 kD carboxv-terminal portion of the heavv chain is referred to as "^ ihe C fiagment oi the H( domain Expression of the C Iragment ot C hoiulinum tvpe 1 toxin in heterologous hosts (e g L coh) has not been previously reported
The native C fiagment of the C botulinum serotvpe F toxin (BotE) gene was cloned and inserted into expression vectors to tacilitate expression ot the recombinant BotE protein in / coh 1 his example involved PCR amplification of the gene cloning, and construction of 0 expression vectors
Ihe BotL serotvpe gene was isolated using PCR as described for the BotA serotvpe gene in Fxample 28 Ihe C botulinum tvpe F strain was obtained trom the American Type C ultuic C ollection (ATCC 17786 strain 2231) The lollowing primer pair was used in the PCR amplification 3 -C GCCA IGGCT C ITTCTTCTTAT ACΛGΛTGΛ1-3 (5 primer 5 engineered I site underlined) (SEQ ID NO 57) and -CiC \G TI77 ITTTTICTIGCC \ FC CAT G-3 (3 primer engineered Hindi 11 site undo lined native gene termination codon italicized) (SFQ ID NO 58) The PCR product was inserted into pC Rscπpt as described in 1 xample 28 The iesulting pC Rscπpt BotF clone was confirmed bv restriction digestion, as well as. by obtaining the sequence of approximatelv 300 0 bases located at the 5 end of the C fiagment coding region using standard DNA sequencing methods Ihe resulting BotI sequence was identical to that of the published C botulinum tvpe I toxin sequence [Whelan et al (1992). supia]
The
Figure imgf000205_0001
Iragment from a pCRscπpt Botl recombinant vvas cloned into pLTHisb vector as described for BotA C fragment in Example 28 The resulting construct "v was to med pHisBotE pHisBotE expresses the BotL gene under the control of the T7 lac promoter and the resulting protem contains an N-terminal lOXHis-tag affinity lag
The pHisBotE expression construct was transformed into BL21(DL3) pLysS competent cells and 1 liter cultures were grown induced and his-tagged proteins weie purified utilizing a NiNTA iesrn (eluted in low pH elution buffer) as described in Example 28 Total soluble 0 and punfied proteins were resolved bv SDS-PAGF and detected bv Coomassie staining The lesults aie shown in Figure 38
In Tigure 38, lane 1 contains broad range MW markeis (BioRad) lane 2 contains a total protein extract, lane 3 contains a soluble protem extract lane 4 contains proteins piesent
- 202 in the flow through from the NiNTA column (this sample was not diluted prior to loading and therefore represents a load 5X that of the load applied for the total and soluble extracts in lanes 2 and 3 ); lane 5 contains proteins eluted from the NiNTA column; lane 6 contains protein eluted from a NiNTA column which had been stored at -20°C for 1 year. The pHisBotE protein was expressed at moderate levels ( 7 mg/liter) as a totally soluble protein. T he purified protein migrated as a single band of the predicted MW.
Western blot hybridization utilizing a chicken anti-C botulinum se otype E toxoid primary antibody (generated by immunization of hens as described in Example 3 using C. botulinum serotype E toxoid ) was also performed on the total, soluble and purified BotE proteins. Samples of BotA and BotB C fragments were al.so included on the gels to facilitate
MW and immunogenicity comparisons. Strong immunoreactivity was detected using the anti-C. hoiulinum type E toxoid antibody only with the BotE protein.
Fhese results demonstrate that the native BotE gene sequences can be expressed as a soluble his-tagged protein in E. coli and purified by metal-cheiation affinity chromatography.
EXAMPLE 42 Generation Of Neutralizing Antibodies Using T he Recombinant pHisBotE Protein
The ability of the purified pHisBotE protein to generate neutralizing antibodies was examined. Nine BALBc mice were immunized with BotE protein ( purified as described in
Ex. 41 ) using Gerbu GMDP adjuvant (CC Biotech). The low pH elution was mixed with Gerbu adjuvant and used to immunize mice. Each mouse received a subcutaneous injection of 100 μl antigen/adjuvant mix (35 μg antigen + 1 μg adjuvant ) on day 0. Mice were subcutaneously boosted as above on day 14 and bled on day 28. Mice were subsequently boosted and bled on day 70.
Anti-C. botulinum serotype E toxoid titers were determined in day 28 serum from individual mice from each group using the ELISA protocol outlined in Example 29 with the exception that the plates were coated with C botulinum serotype E toxoid. and the primary antibody was a chicken anti-C. botulinum serotype E toxoid. Seroconversion [relative to control mice immunized with the p6xI IisBotA antigen (Fix. 29)] was observed with all 9 mice immunized with the purified pFIisBotE protein.
The ability of the anti-BotE antibodies to neutralize native ( ' hoiulinum type E toxin was tested in a mouse-C". hoiulinum neutralization model using pooled mouse serum (see Ex. 23b) The LI , of purified C" botulinum type E toxin complex (Dr. Eric Johnson. University ot Wisconsin. Madison) was determined by a intraperitoneal (IP) method [Schantz and Kautler ( 1978). supra] using 18-22 g female ICR mice. The amount of neutralizing antibodies present in the serum of the immunized mice was determined using serum antibody titrations. The v arious serum dilutions (0 01 ml) were mixed with 5 LD , units of C hoiulinum type F toxin and the mixtures were injected IP into mice The neutralizations were performed in duplicate I he mice were then observed for signs of botulism for 4 days. Undiluted serum from day 28 did not protect, while undiluted. 1 /10 diluted and 1 /100 diluted day 70 serum protected ( 1005 ot animals) while 1 /1000 diluted day 70 serum did not. This corresponds to a neutralization titei ol 50-500 lU/ml.
I hese results demonstrate that seroconversion occurred and neutralizing antibodies were induced w hen the recombinant BotE- protem was utilized as the immunogen.
EXAMPLE 43 Construction Of Vectors I o Facilitate Expression
Of His- l agged BotE Protein In Fermentation Cultures
\ number of expression vectors were constructed to facilitate the expression of i ecombinant BotE protem in large scale fermentation culture T hese constructs v aried as to the str ength of the promoter utilized ( F7 or T71ac) and the presence ol repressor elements
( laclq ) on the plasmid I he resulting constructs varied in the level of expression achieved and in plasmid stability w hich facilitated the selection of a optimal expression system toi la mentation scaleup This example inv olved a) construction of BotE expression v ectors and b) determination of expression levels in small scale cultures
a) Construction Of BotE Expression Vectors
The BotE expression vectors created for fermentation culture were engineered to utilize the kanamvcin rather than the ampiciilin resistance gene, and contained either the T7 or F7lac pi molei . w ith or without the laclq gene for the reasons outlined in Example 30 I n all cases, the
Figure imgf000207_0001
expressed by the various expression vectors is ihe pHisBotE protein described in E.xample 4 1 . w ith the only differences between clones bong the alteration ol various regulatory elements Using the designations outlined below, the pHisBotE clone ( T.x 41 ) is equivalent to pFIisBotE amp r7Iac. i) Construction Of pHisBotE kan laclq T7Iac pHisBotE kan laclq T71ac was constructed by inserting the ΛΛαl/Hwdlll fragment of pHisBotE which contains the BotE gene sequences into ΛTwI/H/willl-cleaved pFT 24a vector Proper construction was confirmed by restriction digestion
ii) Construction Of pHisBotE kan T7 pI IisBotL kan 1 7 was constructed by ligating the BotI -containing XballSapl fragment of pΗisBotl kan laclqT71ac to the 1 7 promoter-containing XballSapl fragment of pf T23a Propei construction was confirmed by restriction digestion
iii) Construction Of pHisBotE kan lac!qT7 pIlisBot! kan ladq 7 was constructed by insei t g the Bgll llHindlll Iiagment f rom pHisBotE kan 1 7 which contains the BotF gene sequences into # /H////"dI II-cleav ed pLT24 v eetoi Pi per construction was confirmed bv restriction digestion
I s b) Determination Of BotE Expression Levels In Small Scale
C ultures
I he thi ee BotF kan expression vectors described above were transformed into B121 (DL3 ) competent cells and 50 ml (2XYT + 40 μg/ml kan) cultures were grown and induced w ith IT PG as described in Example 28 Total and soluble protem extracts trom befoi e and af ter induction made as described in I xample 28 I he total and soluble extracts were i csolv ed on a 12 5% SDS-PAGE gel, and his-tagged proteins wei e detected on a Western blot utilizing the NiNT A-alkalinc phosphatase conjugate as described in Fxample 3 1 (c)( ιu ) Fhe results showed that all three BotF cell lines expressed his-tagged proteins of the predicted MW lor the BotE protem upon induction The results also demonstrated that the two consti ucts that contained the 1 7 promoter expressed the BotI protem before induction, while the T71ac promotei construct did not Upon induction, the T 7 promoter- containing constructs induced to highei levels than the T71ac-contaιnιng construct w ith the pl lisBotl kan Iaclq l 7/B121 (DL3) cells accumulating the maximal lev els ol BotI proton EXAMPLE 44
Expression And Purification Of pHisBotE From Fermentation Cultures
Based on the small scale inductions performed in Exampie 43. the pHisBotE kan laclq F7/BI21 (DE3 ) strain was selected for fermentation scaieup. This example involved the la mentation and purification of recombinant BotE C fragment protein
A fermentation with the pHisBotE kan laclq T7/B121 (DE3) strain was performed as described in Example 31 The fermentation culture was induced 2 hrs post start of the glucose feed with 4 gm IP TG (final concentration = 1 6 mM) The OD,,„„ was 42 at time of induction, then 46.5. 48. 53 and 54 at 1 -4 hrs post induction. Viable colony counts decreased
Irom 0-4 hr induction [ 13 1. 4 (28). 7 (3), 7, 8; dilution 3 utilized 6 μl of dilution 2 cells, bracketed colonies are microcolonies] Ail (32/32) colonies scored at the time of induction t eiatned the BotE plasmid ( kan resistant) and no colonies at induction grew on I PTG+Kan plates ( no mutations detected) These results were indicative of strong promoter induction. since colonv v iability reduced after induction, and the culture stopped growing during lermentation ( stopped at 54 ODwl /ml )
Total and soluble extracts were resolved on a 12.5% SDS-PAGE gel and total protein vvas detected by staining with Coomassie blue Fhe results are shown in Figure 39
I n F igure 39. lane 1 contains total protein from a pHisBotA kan T7 lac/B121 (DE3 ) pLv sS fermentation ( Ex. 24) Lanes 2-9 contain extracts prepared from the abov e pHisBotE kan laclq I 7/BI21 (DE3) fermentation, lanes 2- 4 contain total protein extracts piepared at 0. 1 and 2 houi s post-induction, respectively Lane 5 contains a soluble proton extract prepared at 2 hours post-induction Lanes 6 and 7 contain total and soluble extracts prepared at 3 hours |X)st-ιnductιon. respectively Lanes 8 and 9 contain total and soluble extracts prepared at 4 hours post-induction, respectivelv Lane 10 contains broad range MW markers (BioRad)
T he results shown in F igure 39 demonstrate that moderate level induction of totally soluble Bot E protein was observ ed, increasing from 1 to 4 hrs post induction (no expression vvas detected in uninduced cells) From a 2 liter fermentation harvest a 155 gm ( et vvt) cell pellet vvas obtained and used to make a PEI-clarified lysate ( 1 liter m CRB. pl l 6.8 ) The lysate was applied to a large scale IDA column and 200 mg of BotE protein, which was found to be greater than 95% pure (as |udged by visual inspection of a Coomassie stained SDS-PAGE gel), was recovered. This represents 2.5% of the total soluble cellular proton (assuming a PEI Ivsate having a concentration of 8 mg proton/ml) and corresponds to a y ield ol 100 mg BotE pioton/liter ol fermentation culture
The above results demonstrate that high lev els of the iecombinant BotF protein can be expressed and purified f i om fermentation cultures s
EXAMPLE 45
Removal Of Imidazoie Fiom Purified Recombinant BotF Piote Pieparations
I he expression ol i ecombinant BotE protein, unlike the Bot A. and BotB pioteins did
10 not requite the presence ol tolding chaperones to maintain solubility during scale up Λ size exclusion chromatographv step was included however to remove the imidazoie trom the sample and exchange the IDA elution buffer for one consistent w ith the BotA antigen
\ Sephacrv l S- 1 00 HR (S- 100 Pharmacia) column was poured ( 2 5 cm x 24 cm bed v olume 1 10 ml) Under these conditions, the BotF proton should be retained bv the beads
I 3 to a lesser degree than the smaller imidazoie therefore the Boll piote should elute trom the column bef ore the imidazoie I he column vvas equilibrated in a but lei consisting ol 50 mM sodium phosphate 0 5 M NaC l and 10% glvcerol (all reagents ti om Mallinkrodt ) I lv e millilito s ol the IDA-purified BotL protem ( Lx 44 ) was filtered through a 0 43 μ sv πnge filter and applied to the S- 100 column and equilibration buf fei was pumped through the
20 column at a flow tate ot I ml/minute Eluted proteins were monitored bv absorbance at 280 nm and collected either manuallv or with a fraction collectoi Appropriate tubes were pooled il necessaiv and the protein was quantitated bv absor bance at 280 nm and/oi BC \ piotein assav Fhe isolated peaks were then analvzed by native and/oi SDS-P AG1 lo identify the pιotem( s) and ev aluate the puπtv
25 I igure 40 provides a representative chromatogram showi u the purification ol
IDA-punlled BotI on the S- 100 column Even though folding chaperones were not ov er-expressed with this antigen a small amount ot protem eluted at a time consistent with the tolding chaperones expressed w ith BotA and BotB pi otems ( Gi o) ( see the f u st peak) The second peak in the chromatogi am contained the BotI proton and the thud peak was
30 presumably imidazoie I his pi esumed imidazoie peak was isolated in comparable lev els in
IDA-punlled BotA and BotB protem preparations as well These results demonstrate that size exclusion chromatography can be used to remove imidazoie and traces of contaminating high molecular weight proteins from IDA-purified BotF protein preparations
Fhe S- 100-puπfιed BotE proton was tested for endotoxin contamination using the - 5 L AI assay as descπbed in Example 24 I his preparation vvas found to contain 64 to 128
I Umig recombinant proton and is therefore substantially tree ot endotoxin
The S- 100 purified BotE was mixed with purified preparations of BotA and BotB proteins and used to immunize mice. 5 μg of each Bot proton was used per immunization and alum was included as an adjuvant After two immunizations with this t valent vaccine. 10 the immunized mice were challanged with (' hoiulinum toxin I he immunized mice contained neutralizing antibodies sufficient to neutralize between 100.000 to 1.000.000 LD ) of othei toxin A or toxm B and between 1.000 to 10.000 1 D,„ ol toxin 1 I he tiler ol neiiti alizmg antibodies directed against toxin E would be expected to increase following subsequent boosts w ith the v accine I hese lesults demonstrate that a triv alent v accine I ^ containing i ecombinant BotA. BotB and BotE proteins provokes neutralizing antibodies
EXAMPLE 46
Expression Ol I he C I ragment Of T he ( ' hoiulinum Serotype C Toxin Gene And Generation Of Neutralizing Antibodies 20
Hie C hoiulinum ty pe C l neurotoxin gene has been cloned and sequenced | imura ei al ( 1990) Biochem Biophv s Res C omm 171 1 304] 1 he nucleotide sequence of the toxin gene dei iv ed I rom the C botulinum t} pe C strain C-Stockhoim is available li om the LMBI /GenBank sequence data banks under the accession number D902 I 0. the nucleotide 25 sequence ot the coding region is Iisted m SEQ ID NO 59 The ammo acid sequence ot the C hoiulinum t} pe C 1 neurotoxin derived trom this strain is iisted in SEQ ID NO 60
The DNA sequence encoding the native C botulinum seron pe C l C fragment gene derived from the C-Stockholm strain can be expressed using the pF I Hisb vector, the resulting coding legion is listed in SEQ ID NO 61 and the corresponding amino actd sequence is Iisted 30 in SFQ ID NO 62 The C fragment region from any stiain of ( ' hoiulinum seiotype C can be amplified and expressed using the approach illustrated below using the C Iiagment derived Irom ( botulinum type C C-Stockholm strain Expression ot the C fragment of C botulinum type C I toxin in heterologous hosts (e L coh) has not been prev iously reported The C fragment of the C botulinum serotype Cl (BotCl) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene. A number of C hoiulinum serotype C strains (expressing either or both Cl and C2 toxin) aic available from the ATCC [eg.2220 (ATCC 17782).2239 (ATCC 17783).2223 (AICC 17784. a type C-β strain. C-β strains produce C2 toxin).662 (ATCC 17849. a type
C-(x strain. C'-α strains produce mainly CT toxin and a small amount of C2 toxin).2021 (ATCC 17850. a type C-u strain) and VPI 3803 (AICC 25766)) .Alternatively, other type C strains may be employed for the isolation of sequences encoding the C fragment of (' botulinum serotype C toxin. The lollowing primer pair is used to amplify the BotC gene 5 -CGCCATGGC
TTTAFTAAAAGATAFAATTAATG-3' [5' primer, engineered .WI site underlined (SEQ ID NO 63)] and 5'-G AAGCTT7T ITCACTT ACΛGGTAC ΛΛAACC-3" |3" primer, engineered Hindlll site underlined, native gene termination codon italicized (SEQ ID NO 64)] I ollowing PCR amplification, the PCR product is inserted into the pCRscnpt vector and then the 15 kb Iragment is cloned into pETHisb vector as described lor BotA C fragment gene in Example 28 Fhe resulting construct is termed pHisBotC Proper construction is confirmed by DNA sequencing ot the BotC sequences contained within pHisBotC pHisBotC expresses the BotC gene sequences under the transcπptional control ot the T7 lac promoter and the resulting protein contains an N-terminal lOXHis-tag affinity tag Ihe pHisBotC expression construct is transformed into BI 21(DE3) pi vsS competent cells and 1 liter cultures are grown, induced and his-tagged proteins aie purified utilizing a NiNlΛ resin (eluted in 250 mM imidazoie.20% glycerol) as described in Fxample 28 l tal. soluble and purified protons are resolved by SDS-PAGF and detected by ( oomassie staining and Western blot hybridization utilizing a Ni-N TA-alkal e phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in Example 31(c)(uι) This analysis permits the determination of expression levels ol the pHisBotC proton (/ e . number ot mg/hter expressed as a soluble proton) The purified BotC protein will migrate as a single band of the predicted M (ιe. 50kD)
Fhe level of expression of the pHisBotC proton may be modified (increased) by substitution ol the F7 promoter lor the I 7lac promoter, or by inclusion ot the laclq gene on the expiession plasmid. and plasmid expressed in BE2KDF3) cell lines m fermentation cultures as desciibed in E.xample 30 If only very low levels (/ e . less than 05%) of soluble pHisBotC protein are expressed using the above expression systems, the pFhsBotC construct may be co-expressed with pACYCGro construct as described in Example 32. In this case, the recombinant BotC protein may co-purify with the folding chaperones. The contaminating chaperones may be removed as described in Example 34. Preparations of purified pHisBotC protein are tested for endotoxin contamination using the LAL assay as described in Example 24.
The purifed pHisBotC protein is used to generate neutralizing antibodies. BALBc mice are immunized with the BotC protein using Gerbu GMDP adjuvant (CC Biotech) as described in Example 36. The ability of the anti-BotC antibodies to neutralize native C. botulinum type C toxin is demonstrated using the mouse-C. botulinum neutralization model described in Example 36.
EXAMPLE 47
Expression Of The C Fragment Of The C. botulinum Serotype D Toxin Gene And Generation Of Neutralizing Antibodies
The ( ' botulinum type D neurotoxin gene has been cloned and sequenced [Sunagawa et al. ( 1992) J. Vet. Med. Sci. 54:905 and Binz et al. ( 1990) Nucleic Acids Res. 1 8:5556]. The nucleotide sequence of the toxin gene derived from the CB 16 strain is available from the EMBL/GenBank sequence data banks under the accession number S49407: the nucleotide sequence of the coding region is listed in SEQ ID NO:65. The amino acid sequence of the C. hoiulinum type D neurotoxin derived from the CB 16 strain is listed in SEQ ID NO:66.
Fhe DNA sequence encoding the native C. botulinum serotype D C fragment gene derived from a BotD expressing strain can be expressed using the pETHisb vector: the resulting coding region is Iisted in SEQ ID NO:67 and the corresponding amino acid sequence is Iisted in SEQ ID NO:68. T he C fragment region from any strain of ( '. botulinum serotype
D can be amplified and expressed using the approach illustrated below using the C fragment derived from ( ' botulinum type D CB 16 strain. Expression of the C fragment of C. hoiulinum type D toxin in heterologous hosts (e.g. , E. coli) has not been previously reported. Fhe C fragment of the C. hoiulinum serotype D (BotD) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene.
A number of C. hoiulinum type D strains are available from the ATCC [e.g. , ATCC 9633. 2023 (ATCC 17851 ). and VPI 5995 (ATCC 27517)]. The following primer pair is used to amplify the BotD gene 5'-CGCCATGGC TTTAT TAAAAGATATAATTAATG-3* [5' primer, engineered Ncol site underlined (SEQ ID NO 63)] and 5'-GCAAGCTT7T CTC1 ACCCAT CC I GG ATCCCT-3* [V primer, engineered Hindlll site underlined, native gene termination codon italicized (SEQ ID NO 69)] I ollowmg PCR amplification, the PCR product is inserted into the pCRscnpt vector and then the I 5 kb fragment is cloned into pETHisb vector as described foi BotA C fragment gene in F xample 28 The resulting construct is termed pHisBotD pFhsBotD expresses the BotD gene sequences under the transcπptionai control ol the T7 lac pi omoter and the resulting protein contains an N-terminal l OXHis-tag alfinity tag I he pHisBotD expression construct is transformed into BL21 (DE3) pi ysS competent cells and 1 litei cultures aie grown, induced and his-tagged protons are punfied utilizing a NiN TA resin as described in Example 28 Total, soluble and purified pioteins are resolv ed by SDS-PAGE and detected bv C oomassie staining and Western blot hy bridization utilizing a Ni-NTΛ- alkahne phosphatase conjugate (Qiagen) which recognizes his-tagged protons as described in Example 3 | (c)( uι) This analysis permits the determination ot expression lev els of the pHisBotD protem (/ e . number of mg/liter expressed as a soluble protein ) I he purified BotD protein w ill migrate as a single band ol the predicted MW ( / c 50kD)
I he lev el ol expression ot the pHisBotD proton mav be modified ( increased) bv substitution of the T7 promoter lor the T7lac promoter, or by inclusion of the laclq gene on the expression plasmid. and plasmid expressed in BI 21 (DE3 ) cell lines in fermentation cultures as described in Example 30 II onlv verv low lev els (/ c . less than about 0 5%) of soluble pHisBotD protein are expressed using the above expression sy stems the pHisBotD construct mav be co-expressed w ith pACYCGro construct as described in F xample 32 In this case, the recombinant BotD proton may co-puπ with the folding chaperones T he contaminating chaperones may be removed as described in I xample 34 Preparations ot purified pHisBotD protem are tested tor endotoxin contamination using the LΛL assay as desci ibed in 1 xample 24
T he punted pHisBotD protein is used to generate neutralizing antibodies BΛLBc mice ai e immunized with the BotD proton using Gerbu GMDP adjuvant (C C Biotech) as described in Example 36 T he ability ot the anti-BotD antibodies to neutralize native ( hoiulinum tv pe D toxin is demonstrated using the mouse-C hoiulinum neutralization model described in I xample 36 EXAMPLE 48
Expression Of The C Fragment Of The C hoiulinum Serotype F Toxin Gene And Generation Of Neutralizing Antibodies
The C botulinum type F neurotoxin gene has been cloned and sequenced [East et al
{ 1992) FEMS Microbiol Lett 96225] The nucleotide sequence of the toxin gene derived liom the 2021 stiain (ATCC 23387) is available from the EMBL/GenBank sequence data banks under the accession number M92906. the nucleotide sequence of the coding region is listed in SFQ ID NO 70 The amino acid sequence of the C hoiulinum type 1 neurotoxin derived liom the 202F strain is listed in SLQ ID NO 71
Ihe DNA sequence encoding the native (' botulinum seiotype I C Iiagment gene derived irom the 202F strain can be expres.sed using the pE I Hisb vector, the resulting coding region is Iisted m SFQ ID NO 72 and the corresponding ammo acid sequence is listed in SLQ II) NO 73 I he C fragment region trom any strain oϊ C hoiulinum serotype F can be amplified and expressed using the approach illustrated below using the C Iiagment deiived liom ( hoiulinum type F 2021 strain Fxpression of the C fragment of C hoiulinum type F toxm in heterologous hosts (e E coh) has not been previously reported
Ihe C Iiagment of the C hoiulinum serotype 1 (BotI ) toxin gene is cloned using the protocols and conditions described in F ample 28 for the isolation oi the native BotA gene Ihe ( hoiulinum tvpe I 202T strain is obtained fiom the American Ivpe C ulture Collection
(AICC 23387)
Figure imgf000215_0001
. sequences encoding the BotF toxin may be i.solated Irom any BotI expiessmg strain [e g VPI 4404 (A FCC 25764) VPI 2382 (AICC 27321 ) and Langeland (AICC 35415)]
Ihe following primer pair is used to amplify the BotF gene s'-CG CA I GGC IΛITC'IΛAl TAT Al ATTTTAA I AG-3" [5' primer, engineered WI site underlined (SFQ
ID NO 74)| and 5'-GCAAGCT TCA TTCTTTCCA I CCATTCTC-3" [3" primer, engineered Hindlll site underlined, native gene termination codon italicized (SFQ ID NO 75)] Following PCR amplification, the PCR product is inserted into the pCRscnpt veetoi and then the 15 kb fragment is cloned into pETHisb vector as described for BotA C Iragment gene Example 28 The resulting construct is termed pHisBotF pIIisBotF expresses the BotF gene sequences under the transcnptional control of the 17 lac piomoter and the lesulting protein contains an N-terminal IOXHis-tag affinity tag The pHisBotl expiession construct is tianstormed into BL21(DP3) pL} sS competent ceils and 1 liter cultuies are grown, induced and his-tagged proteins are purified utilizing a NiNTA resin as described in Example 28 Total, soluble and purified proteins are resolved by SDS-PAGE and detected b} Coomassie staining and Western blot hybridization utilizing a Ni-NTA- alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in Example 3 1 (c)(ιιι ) This analysrs permits the determination ol expression levels of the pHisBotF protein (/ e . number of mg/liter expressed as a soluble protein ) T he punfied BotF protem will migrate as a single band of the predicted MW (/ e 5()kD)
T he level ol expression of the pHisBotF proton may be modified (increased) by substitution of the T 7 promoter for the T71ac promoter, or bv inclusion of the laclq gene on the expression plasmid. and plasmid expressed m BL21 (DE3) cell lines in f ermentation cultures as described in Example 30 If only very low levels (; e less than about 0 5%) of soluble pHisBotF protein are expressed using the above expression systems, the pI IisBotl construct may be co-expressed w ith pACYCGro construct as desci ibed in Fxample 32 I n this case, the iecombinant BotF protein may co-puπfy with the folding chaperones T he contaminating chapeiones may be removed as described in Fxample 34 Pieparations ol purified pl iisBotl protem are tested foi endotoxin contamination using the LAL assay as described in 1 xample 24
I he punted pHisBotF proton is used to generate neutralizing antibodies BALBc mice ai e immunized with the BotI proton using Gerbu GMDP adjuvant (CC Biotech) as desci ibed in 1 xample 36 The ability of the anti-Botl antibodies to neutiahze nativ e ( hoiulinum ty pe 1 toxm is demonstrated using the mouse-( hoiulinum neutralization model described m E.xample 36
EXAMPLE 49 Expression Of The C Fragment Of The ( hoiulinum
Serotype G Toxin Gene And Generation Of Neutralizing Antibodies
T he ( hoiulinum ty pe G neurotoxin gene has been cloned and sequenced (Campbell el al ( 1993) Biochimica et Biophysica Acta 1216 487 and Binz et al ( 1990) Nucleic Acids Res 1 8 5556 ] The nucleotide sequence of the toxin gene deπved tiom the 1 1 /30 strain (NCFB
3012 ) is av ailable Irom the F MBL/GenBank sequence data banks undei the accession number X74162. the nucleotide sequence ot the coding region is listed in SEQ ID NO 76 The amino acid sequence of the C botulinum type Ci neurotoxin derived from this strain is listed in SEQ ID NO 77
The DNA sequence encoding the native C botulinum serotype G C fragment gene derived from the 1 1 3/30 strain can be expressed using the pETFlisb vector, the resulting coding region is listed in SEQ ID NO 78 and the corresponding amino acid sequence is listed in SFQ ID NO 79 The C Iragment region from any strain of C
Figure imgf000217_0001
serotype G can be amplified and expressed using the approach illustrated below using the C Iiagment derived liom ( hoiulinum type G I 13/30 stiain Expression ot the C fragment of C hoiulinum type G toxin in heterologous hosts (e g L co ) has not been previously reported I he C fiagment of the C botulinum serotype G (BotG) toxin gene is cloned using the protocols and conditions described in Example 28 for the isolation of the native BotA gene T he C hoiulinum type G 1 13/30 stiain is obtained from the NCFB. Fhe following primer pair is used to amplify the BotC} gene 5"-CGCCA 1 GGCTGAC ACAA TTTT AA TACA AG I -3" [5 ' primer, engineered Xcol site underlined (SEQ ID NO 80)] and 5 --GCCTCGAG7TT I FCTGTCCA I CCTTCAT CCAC-3" [3" primer, engineered Xhol site underlined, native gene termination codon italicized (SEQ ID NO 8 1 )] I ollow ing PCR amplification, the PCR product is inserted into the pCRscnpt vector and then the 1 5 kb iragment is cloned into pETHisb v ector as described for BotA C fragment gene in Example 28 with the exception that the sequences encoding BotG aie excised from the pCRscnpt v ector by digestion with Xcol and Xhol and the Ncol site is blunted (the BotG sequences contain an internal Hindlll site) This W>l(fiHed)/Λ7wI fragment is then ligated to the pi πiisb v eetoi which has been digested w ith Λ7?cl and Sail and the Mid site is blunted Fhe resulting construct is termed pHisBotG pHisBotG expresses the BotG gene sequences under the transcπptional control of the 1 7 lac promotei and the resulting protein contains an N-terminal l OXHis-tag affinity tag. The pHisBotG expression construct is transformed into BL2HDE3) pLysS competent cells and 1 liter cultures are grown, induced and his-tagged proteins aie purified utilizing a NiNTA resin as described in Example 28 l otal. soluble and purified proteins are resolved by SDS-PAGE and detected by C oomassie staining and Western blot hybridization utilizing a Ni-N I Λ- alkaline phosphatase conjugate (Qiagen) which recognizes his-tagged proteins as described in
1 xample 3 1 (c)(ιιι) This analysis pei mits the determination of expression levels of the pHisBotG protein ( / e . number of mg/hter expressed as a soluble protem) I he purified BotG proton will migrate as a single band of the predicted MW (/ c . ~-50kD) The level of expression of the pHisBotG protein may be modified (increased) by substitution of the T7 promoter for the T71ac promoter, or by inclusion oϊ the laclq gene on the expression plasmid. and plasmid expressed in BL21 (DE3) cell lines in fermentation cultures as descπbed in Example 30 If only very low levels (i e . less than about 0 5%) of soluble pHisBotG protem are expressed using the above expression systems, the pHisBotG construct may be co-expressed with pACYCGro construct as descπbed in I xample 32 In this case, the recombinant BotG protein may co-puπfv with the tolding chaperones The contaminating chaperones may be removed as descπbed in Example 34 Pi eparations ot purified pHisBotG protem are tested for endotoxin contamination using the LAL assay as descπbed in I xample 24
I he puπfed pHisBotG protein is used to generate neutralizing antibodies BALBc mice ai e immunized w ith the BotG proton using Gerbu GMDP adjuvant ( CC Biotech) as described in F xample 36 The ability ot the anti-BotG antibodies to neutralize native ( hoiulinum tv pe G toxin is demonstrated using the mouse-C hoiulinum neutiahzation model descπbed in Lxample 36
EXAMPLE 50
F xpression Of Recombinant Botulinal l oxin Proteins In Fucaryotic Host C ells
Recombinant botulinal C Iragment proteins may be expressed in eucai v tic host cells, such as v east and insect cells
a) Expression In V east
Botulinal C f ragments derived f rom serotypes A. B. C D E. F and CJ may be expressed in yeast cells using a v ariety of commercially available v ectors 1 or example, the pPIC3K and pPIC9K expression vectors ( Invitrogen) may be emploved (oi expression in the methyloti ophic yeast. Piclua pasloi is When the pPIC3K veetoi is employed expression of the botulinal C Iragment protein will be intraceliulai When the pPlC3K vector is emploved. the botulinal C f iagment proton w ill be secreted (the alpha factor seciction signal is provided on the pPIC9K vector)
DNA sequences encoding the desired C f ragment is inset ted into these v ectors using techniques known to the art Briefly, the desired botulinal expression cassette (including sequences encoding the his-tag. described in the preceding examples) is amplified using the PCR in conjunction with primers that incorporate unique restriction sites at the termini of the amplified fragment. Suitable restriction enzyme sites include SnaBl. EcoRl. Avrll and Noll. When the botulinal C fragment is to be expressed using the pPIC3K vector, the initiator methionine (ATG) is provided by the desired Bot gene sequence and a Kozak consensus
• sequence is engineered upstream of the ATG (e.g., ACCATGG).
Fhe amplified restriction fragment containing the botulinal C fragment gene is then cloned into the desired expression vector. Recombinant clones are integrated into the Pichia pastoπs genome and recombinant protein expression is induced using methanol following the manufacturer' s instructions (Invitrogen Pichia expression kit manual).
( '. botulinum genes are A/T rich and contain multiple sequences that are similar to yeast transcriptional termination signals (e.g., TTTTTATA). If premature transcription termination is observed when the botulinal C fragment genes are expres.sed in yeast, the transcription termination signals present in the C fragment genes can be removed by either site directed mutagenesis (utilizing the pALTER system: Promega) or by construction of synthetic genes utilizing overlapping synthetic primers.
T he botulinal C fragment genes may be expressed in other yeast cells using other commercially available vectors [e.g.. using the pYES2 vector (Invitrogen) and S cerevisiae cells ( Invitrogen)].
20 b) Expression In Insect Cells
Botulinal C fragments derived from serotypes A. B. C. D. E. F and G may be expressed in insect cells using a variety of commercially available vectors. For example, the pBlueBac4 transfer vector ( Invitrogen) may be employed for expression in Spodopiera frugiperda (S >) insect cells (baculovirus expression system) (equivalent baculovirus vectors
25 and ho.st celis are avaialble from other vendors, e.g., Pharmingen. San Diego. CA). Botulinal
C fragments contained on NcollHindlll fragments contained within the pHisBotA-G expression constructs (described in the preceding examples) are cloned into the pBlueBac4 vector (digested with Ncol and Hindlll): the Ncol site present on the C fragment constructs overlaps with the start codon of the fusion proteins. In the case of botulinal C fragment
30 clones that contain internal ITindlll sites (e ., using the BotG sequences described in Ex. 49). the C fragment gene is contained within a Ncol/Xhol fragment on the pHisBot construct. This Ncol/Xhol fragment is excised from pHisBot and inserted into pBlueBac4 digested with Ncol and Sail. Recombinant baculoviruses are made and the desired recombinant C fragment is expres.sed in Sβ cells using the protocols provided by the manufacturer (Invitrogen MaxBac manual). The resulting constructs will express the pHisBot protein intracellularly (including the N-terminal his-tag) under the control of the polyhedrin promoter. For extracellular secretion of botulinal C fragment proteins, the C fragment sequences from the pHisBot constructs are cloned into the pMelBacB vector (Invitrogen) as described above for the pBlueBac4 vector. When the pMelBacB vector is employed, the his-tagged _botulinai C fragment proteins are secreted (utilizing a vector-encoded honeybee melittin secretion signal) and contain a nine amino acid extension at the N-terminus.
His-tagged botulinal C fragments expressed in yeast or insect cells are purified using metal chelation columns as described in the preceding examples.
From the above it is clear that the present invention provides compositions and methods for the preparation of effective multivalent vaccines against C. botulinum neurotoxin. It is also contemplated that the recombinant botulinal proteins be used for the production of antitoxins. All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: Williams, James A.
Thalley, Bruce S.
(ii) TITLE OF INVENTION: Multivalent Vaccine For Clostridium Botulinum Neurotoxin
(in) NUMBER OF SEQUENCES: 82
(ιv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Medlen & Carroll (B) STREET: 220 Montgomery Street, Suite 2200
(C) CITY: San Francisco
(D) STATE: California
(E) COUNTRY: United States of America
(F) ZIP: 94104
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS -DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30
\ i) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: (C) CLASSIFICATION:
(vi ii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Carroll, Peter G.
(B) REGISTRATION NUMBER: 32,837 (C) REFERENCE/DOCKET NUMBER: OPHD-02959
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 705-8410
(B) TELEFAX: (415) 397-8338
(2) INFORMATION FOR SEQ ID NO : 1 : d) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(il) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GGAAATTTAG CTGCAGCATC TGAC (2) INFORMATION FOR SEQ ID NO : :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :
TCTAGCAAAT TCGCTTGTGT TGAA (2) INFORMATION FOR SEQ ID NO : 3 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :
CTCGCATATA GCATTAGACC 20
(2) INFORMATION FOR SEQ ID NO : 4 : (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : CTATCTAGGC CTAAAGTAT 19
(2) INFORMATION FOR SEQ ID NO : 5 :
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8133 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..8130
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
ATG TCT TTA ATA TCT AAA GAA GAG TTA ATA AAA CTC GCA TAT AGC ATT 48 Met Ser Leu lie Ser Lys Glu Glu Leu lie Lys Leu Ala Tyr Ser lie 1 5 10 15
AGA CCA AGA GAA AAT GAG TAT AAA ACT ATA CTA ACT AAT TTA GAC GAA 96
Arg Pro Arg Glu Asn Glu Tyr Lys Thr lie Leu Thr Asn Leu Asp Glu 20 25 30
TAT AAT AAG TTA ACT ACA AAC AAT AAT GAA AAT AAA TAT TTG CAA TTA 144 Tyr Asn Lyε Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gin Leu 35 40 45 AAA AAA CTA AAT GAA TCA ATT GAT GTT TTT ATG AAT AAA TAT AAA ACT 192 Lys Lys Leu Asn Glu Ser lie Asp Val Phe Met Aεn Lys Tyr Lys Thr 50 55 60
TCA AGC AGA AAT AGA GCA CTC TCT AAT CTA AAA AAA GAT ATA TTA AAA 240 Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp lie Leu Lyε 65 70 75 80
GAA GTA ATT CTT ATT AAA AAT TCC AAT ACA AGC CCT GTA GAA AAA AAT 288 Glu Val He Leu He Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95
TTA CAT TTT GTA TGG ATA GGT GGA GAA GTC AGT GAT ATT GCT CTT GAA 336 Leu His Phe Val Trp He Gly Gly Glu Val Ser Asp He Ala Leu Glu 100 105 110 TAC ATA AAA CAA TGG GCT GAT ATT AAT GCA GAA TAT AAT ATT AAA CTG 384
Tyr He Lys Gin Trp Ala Asp He Asn Ala Glu Tyr Asn He Lys Leu
115 120 125
TGG TAT GAT AGT GAA GCA TTC TTA GTA AAT ACA CTA AAA AAG GCT ATA 432
Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala He 130 135 140
GTT GAA TCT TCT ACC ACT GAA GCA TTA CAG CTA CTA GAG GAA GAG ATT 480
Val Glu Ser Ser Thr Thr Glu Ala Leu Gin Leu Leu Glu Glu Glu He 145 150 155 160
CAA AAT CCT CAA TTT GAT AAT ATG AAA TTT TAC AAA AAA AGG ATG GAA 528
Gin Asn Pro Gin Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu 165 170 175
TTT ATA TAT GAT AGA CAA AAA AGG TTT ATA AAT TAT TAT AAA TCT CAA 576
Phe He Tyr Asp Arg Gin Lys Arg Phe He Asn Tyr Tyr Lys Ser Gin 180 185 190
ATC AAT AAA CCT ACA GTA CCT ACA ATA GAT GAT ATT ATA AAG TCT CAT 624
He Asn Lys Pro Thr Val Pro Thr He Asp Asp He He Lys Ser His
195 200 205 CTA GTA TCT GAA TAT AAT AGA GAT GAA ACT GTA TTA GAA TCA TAT AGA 672
Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg 210 215 220
ACA AAT TCT TTG AGA AAA ATA AAT AGT AAT CAT GGG ATA GAT ATC AGG 720 Thr Asn Ser Leu Arg Lys He Asn Ser Asn His Gly He Asp He Arg 225 230 235 240
GCT AAT AGT TTG TTT ACA GAA CAA GAG TTA TTA AAT ATT TAT AGT CAG 768
Ala Asn Ser Leu Phe Thr Glu Gin Glu Leu Leu Asn He Tyr Ser Gin 245 250 255
GAG TTG TTA AAT CGT GGA AAT TTA GCT GCA GCA TCT GAC ATA GTA AGA 816
Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp He Val Arg 260 265 270
TTA TTA GCC CTA AAA AAT TTT GGC GGA GTA TAT TTA GAT GTT GAT ATG 864
Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met
275 280 285 CTT CCA GGT ATT CAC TCT GAT TTA TTT AAA ACA ATA TCT AGA CCT AGC 912
Leu Pro Gly He His Ser Asp Leu Phe Lys Thr He Ser Arg Pro Ser 290 295 ' 300
TCT ATT GGA CTA GAC CGT TGG GAA ATG ATA AAA TTA GAG GCT ATT ATG 960 Ser He Gly Leu Asp Arg Trp Glu Met He Lys Leu Glu Ala He Met 305 310 315 320
AAG TAT AAA AAA TAT ATA AAT AAT TAT ACA TCA GAA AAC TTT GAT AAA 1008
Lys Tyr Lys Lys Tyr He Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335
CTT GAT CAA CAA TTA AAA GAT AAT TTT AAA CTC ATT ATA GAA AGT AAA 1056
Leu Asp Gin Gin Leu Lys Asp Asn Phe Lys Leu He He Glu Ser Lys 340 345 350
AGT GAA AAA TCT GAG ATA TTT TCT AAA TTA GAA AAT TTA AAT GTA TCT 1104
Ser Glu Lys Ser Glu He Phe Ser Lys Leu Glu Asn Leu Asn Val Ser
355 360 365 GAT CTT GAA ATT AAA ATA GCT TTC GCT TTA GGC AGT GTT ATA AAT CAA 1152
Asp Leu Glu He Lys He Ala Phe Ala Leu Gly Ser Val He Asn Gin 370 375 380 GCC TTG ATA TCA AAA CAA GGT TCA TAT CTT ACT AAC CTA GTA ATA GAA 1200 Ala Leu He Ser Lys Gin Gly Ser Tyr Leu Thr Asn Leu Val He Glu 385 390 395 400 CAA GTA AAA AAT AGA TAT CAA TTT TTA AAC CAA CAC CTT AAC CCA GCC 1248 Gin Val Lys Asn Arg Tyr Gin Phe Leu Asn Gin His Leu Asn Pro Ala 405 410 415
ATA GAG TCT GAT AAT AAC TTC ACA GAT ACT ACT AAA ATT TTT CAT GAT 1296 He Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys He Phe His Asp
420 425 430
TCA TTA TTT AAT TCA GCT ACC GCA GAA AAC TCT ATG TTT TTA ACA AAA 1344 Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys 435 440 445
ATA GCA CCA TAC TTA CAA GTA GGT TTT ATG CCA GAA GCT CGC TCC ACA 1392
He Ala Pro Tyr Leu Gin Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460
ATA AGT TTA AGT GGT CCA GGA GCT TAT GCG TCA GCT TAC TAT GAT TTC 1440
He Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe 465 470 475 480 ATA AAT TTA CAA GAA AAT ACT ATA GAA AAA ACT TTA AAA GCA TCA GAT 1488 He Asn Leu Gin Glu Asn Thr He Glu Lys Thr Leu Lys Ala Ser Asp 485 490 495
TTA ATA GAA TTT AAA TTC CCA GAA AAT AAT CTA TCT CAA TTG ACA GAA 1536 Leu He Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gin Leu Thr Glu
500 505 510
CAA GAA ATA AAT AGT CTA TGG AGC TTT GAT CAA GCA AGT GCA AAA TAT 1584 Gin Glu He Asn Ser Leu Trp Ser Phe Asp Gin Ala Ser Ala Lys Tyr 515 520 525
CAA TTT GAG AAA TAT GTA AGA GAT TAT ACT GGT GGA TCT CTT TCT GAA 1632 Gin Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu 530 535 ' 540
GAC AAT GGG GTA GAC TTT AAT AAA AAT ACT GCC CTC GAC AAA AAC TAT 1680 Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr 545 550 555 560 TTA TTA AAT AAT AAA ATT CCA TCA AAC AAT GTA GAA GAA GCT GGA AGT 1728 Leu Leu Asn Asn Lys He Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575
AAA AAT TAT GTT CAT TAT ATC ATA CAG TTA CAA GGA GAT GAT ATA AGT 1776 Lys Asn Tyr Val His Tyr He He Gin Leu Gin Gly Asp Asp He Ser
580 585 590
TAT GAA GCA ACA TGC AAT TTA TTT TCT AAA AAT CCT AAA AAT AGT ATT 1824 Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser He 595 600 605
ATT ATA CAA CGA AAT ATG AAT GAA AGT GCA AAA AGC TAC TTT TTA AGT 1872 He He Gin Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser 610 615 620
GAT GAT GGA GAA TCT ATT TTA GAA TTA AAT AAA TAT AGG ATA CCT GAA 1920 Asp Asp Gly Glu Ser He Leu Glu Leu Asn Lys Tyr Arg He Pro Glu 625 630 635 640 AGA TTA AAA AAT AAG GAA AAA GTA AAA GTA ACC TTT ATT GGA CAT GGT 1968 Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe He Gly His Gly 645 650 655
7- AAA GAT GAA TTC AAC ACA AGC GAA TTT GCT AGA TTA AGT GTA GAT TCA 2016
Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser
660 665 670
CTT TCC AAT GAG ATA AGT TCA TTT TTA GAT ACC ATA AAA TTA GAT ATA 2064
Leu Ser Asn Glu He Ser Ser Phe Leu Asp Thr He Lys Leu Asp He
675 680 685
TCA CCT AAA AAT GTA GAA GTA AAC TTA CTT GGA TGT AAT ATG TTT AGT 2112
Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser
690 695 700
TAT GAT TTT AAT GTT GAA GAA ACT TAT CCT GGG AAG TTG CTA TTA AGT 2160
Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser
705 710 715 720
ATT ATG GAC AAA ATT ACT TCC ACT TTA CCT GAT GTA AAT AAA AAT TCT 2208
He Met Asp Lys He Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser
725 730 735
ATT ACT ATA GGA GCA AAT CAA TAT GAA GTA AGA ATT AAT AGT GAG GGA 2256
He Thr He Gly Ala Asn Gin Tyr Glu Val Arg He Asn Ser Glu Gly
740 745 750 AGA AAA GAA CTT CTG GCT CAC TCA GGT AAA TGG ATA AAT AAA GAA GAA 2304
Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp He Asn Lys Glu Glu
755 760 765
GCT ATT ATG AGC GAT TTA TCT AGT AAA GAA TAC ATT TTT TTT GAT TCT 2352 Ala He Met Ser Asp Leu Ser Ser Lys Glu Tyr He Phe Phe Asp Ser
770 775 780
ATA GAT AAT AAG CTA AAA GCA AAG TCC AAG AAT ATT CCA GGA TTA GCA 2400
He Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn He Pro Gly Leu Ala 765 790 795 800
TCA ATA TCA GAA GAT ATA AAA ACA TTA TTA CTT GAT GCA AGT GTT AGT 2448
Ser He Ser Glu Asp He Lys Thr Leu Leu Leu Asp Ala Ser Val Ser
805 810 815
CCT GAT ACA AAA TTT ATT TTA AAT AAT CTT AAG CTT AAT ATT GAA TCT 2496
Pro Asp Thr Lys Phe He Leu Asn Asn Leu Lys Leu Asn He Glu Ser
820 825 830 TCT ATT GGG GAT TAC ATT TAT TAT GAA AAA TTA GAG CCT GTT AAA AAT 2544
Ser He Gly Asp Tyr He Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn
835 840 845
ATA ATT CAC AAT TCT ATA GAT GAT TTA ATA GAT GAG TTC AAT CTA CTT 2592 He He His Asn Ser He Asp Asp Leu He Asp Glu Phe Asn Leu Leu
850 855 860
GAA AAT GTA TCT GAT GAA TTA TAT GAA TTA AAA AAA TTA AAT AAT CTA 2640
Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu 865 870 875 880
GAT GAG AAG TAT TTA ATA TCT TTT GAA GAT ATC TCA AAA AAT AAT TCA 2688
Asp Glu Lys Tyr Leu He Ser Phe Glu Asp He Ser Lys Asn Asn Ser
885 890 895
ACT TAC TCT GTA AGA TTT ATT AAC AAA AGT AAT GGT GAG TCA GTT TAT 2736 Thr Tyr Ser Val Arg Phe He Asn Lys Ser Asn Gly Glu Ser Val Tyr 900 905 910 GTA GAA ACA GAA AAA GAA ATT TTT TCA AAA TAT AGC GAA CAT ATT ACA 2784 Val Glu Thr Glu Lys Glu He Phe Ser Lys Tyr Ser Glu His He Thr 915 920 925 AAA GAA ATA AGT ACT ATA AAG AAT AGT ATA ATT ACA GAT GTT AAT GGT 2832 Lys Glu He Ser Thr He Lys Asn Ser He He Thr Asp Val Asn Gly 930 935 940 AAT TTA TTG GAT AAT ATA CAG TTA GAT CAT ACT TCT CAA GTT AAT ACA 2880 Asn Leu Leu Asp Asn He Gin Leu Asp His Thr Ser Gin Val Asn Thr 945 950 955 960
TTA AAC GCA GCA TTC TTT ATT CAA TCA TTA ATA GAT TAT AGT AGC AAT 2928 Leu Asn Ala Ala Phe Phe He Gin Ser Leu He Asp Tyr Ser Ser Asn
965 970 975
AAA GAT GTA CTG AAT GAT TTA AGT ACC TCA GTT AAG GTT CAA CTT TAT 2976 Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gin Leu Tyr 980 985 990
GCT CAA CTA TTT AGT ACA GGT TTA AAT ACT ATA TAT GAC TCT ATC CAA 3024
Ala Gin Leu Phe Ser Thr Gly Leu Asn Thr He Tyr Asp Ser He Gin
995 1000 1005
TTA GTA AAT TTA ATA TCA AAT GCA GTA AAT GAT ACT ATA AAT GTA CTA 3072
Leu Val Asn Leu He Ser Asn Ala Val Asn Asp Thr He Asn Val Leu
1010 1015 1020 CCT ACA ATA ACA GAG GGG ATA CCT ATT GTA TCT ACT ATA TTA GAC GGA 3120 Pro Thr He Thr Glu Gly He Pro He Val Ser Thr He Leu Asp Gly 1025 1030 1035 1040
ATA AAC TTA GGT GCA GCA ATT AAG GAA TTA CTA GAC GAA CAT GAC CCA 3168 He Asn Leu Gly Ala Ala He Lys Glu Leu Leu Asp Glu His Asp Pro
1045 1050 1055
TTA CTA AAA AAA GAA TTA GAA GCT AAG GTG GGT GTT TTA GCA ATA AAT 3216 Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val Leu Ala He Asn 1060 1065 1070
ATG TCA TTA TCT ATA GCT GCA ACT GTA GCT TCA ATT GTT GGA ATA GGT 3264 Met Ser Leu Ser He Ala Ala Thr Val Ala Ser He Val Gly He Gly 1075 1080 1085
GCT GAA GTT ACT ATT TTC TTA TTA CCT ATA GCT GGT ATA TCT GCA GGA 3312 Ala Glu Val Thr He Phe Leu Leu Pro He Ala Gly He Ser Ala Gly 1090 1095 1100 ATA CCT TCA TTA GTT AAT AAT GAA TTA ATA TTG CAT GAT AAG GCA ACT 3360 He Pro Ser Leu Val Asn Asn Glu Leu He Leu His Asp Lys Ala Thr 1105 1110 1115 ' 1120
TCA GTG GTA AAC TAT TTT AAT CAT TTG TCT GAA TCT AAA AAA TAT GGC 3408 Ser Val Val Asn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly
1125 1130 1135
CCT CTT AAA ACA GAA GAT GAT AAA ATT TTA GTT CCT ATT GAT GAT TTA 3456 Pro Leu Lys Thr Glu Asp Asp Lys He Leu Val Pro He Asp Asp Leu 1140 1145 1150
GTA ATA TCA GAA ATA GAT TTT AAT AAT AAT TCG ATA AAA CTA GGA ACA 3504
Val He Ser Glu He Asp Phe Asn Asn Asn Ser He Lys Leu Gly Thr 1155 1160 1165
TGT AAT ATA TTA GCA ATG GAG GGG GGA TCA GGA CAC ACA GTG ACT GGT 3552
Cys Asn He Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly 1170 1175 1180 AAT ATA GAT CAC TTT TTC TCA TCT CCA TCT ATA AGT TCT CAT ATT CCT 3600 Asn He Asp His Phe Phe Ser Ser Pro Ser He Ser Ser His He Pro 1185 1190 1195 1200 TCA TTA TCA ATT TAT TCT GCA ATA GGT ATA GAA ACA GAA AAT CTA GAT 3648 Ser Leu Ser He Tyr Ser Ala He Gly He Glu Thr Glu Asn Leu Asp 1205 1210 1215 TTT TCA AAA AAA ATA ATG ATG TTA CCT AAT GCT CCT TCA AGA GTG TTT 3696 Phe Ser Lys Lys He Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe 1220 1225 1230
TGG TGG GAA ACT GGA GCA GTT CCA GGT TTA AGA TCA TTG GAA AAT GAC 3744 Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245
GGA ACT AGA TTA CTT GAT TCA ATA AGA GAT TTA TAC CCA GGT AAA TTT 3792 Gly Thr Arg Leu Leu Asp Ser He Arg Asp Leu Tyr Pro Gly Lys Phe 1250 1255 1260
TAC TGG AGA TTC TAT GCT TTT TTC GAT TAT GCA ATA ACT ACA TTA AAA 3840 Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala He Thr Thr Leu Lvs 1265 1270 1275 1280
CCA GTT TAT GAA GAC ACT AAT ATT AAA ATT AAA CTA GAT AAA GAT ACT 3888 Pro Val Tyr Glu Asp Thr Asn He Lys He Lys Leu Asp Lys Asp Thr 1285 1290 1295 AGA AAC TTC ATA ATG CCA ACT ATA ACT ACT AAC GAA ATT AGA AAC AAA 3936 Arg Asn Phe He Met Pro Thr He Thr Thr Asn Glu He Arg Asn Lys 1300 1305 1310
TTA TCT TAT TCA TTT GAT GGA GCA GGA GGA ACT TAC TCT TTA TTA TTA 3984 Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu 1315 1320 ' 1325
TCT TCA TAT CCA ATA TCA ACG AAT ATA AAT TTA TCT AAA GAT GAT TTA 4032 Ser Ser Tyr Pro He Ser Thr Asn He Asn Leu Ser Lys Asp Asp Leu 1330 ' 1335 1340
TGG ATA TTT AAT ATT GAT AAT GAA GTA AGA GAA ATA TCT ATA GAA AAT 4080
Trp He Phe Asn He Asp Asn Glu Val Arg Glu He Ser He Glu Asn 1345 1350 1355 1360
GGT ACT ATT AAA AAA GGA AAG TTA ATA AAA GAT GTT TTA AGT AAA ATT 4128
Gly Thr He Lys Lys Gly Lys Leu He Lys Asp Val Leu Ser Lys He 1365 1370 1375 GAT ATA AAT AAA AAT AAA CTT ATT ATA GGC AAT CAA ACA ATA GAT TTT 4176 Asp lie Asn Lys Asn Lys Leu He He Gly Asn Gin Thr He Asp Phe 1380 1385 1390
TCA GGC GAT ATA GAT AAT AAA GAT AGA TAT ATA TTC TTG ACT TGT GAG 4224 Ser Gly Asp He Asp Asn Lys Asp Arg Tyr He Phe Leu Thr Cys Glu 1395 ' 1400 1405
TTA GAT GAT AAA ATT AGT TTA ATA ATA GAA ATA AAT CTT GTT GCA AAA 4272 Leu Asp Asp Lys He Ser Leu He He Glu He Asn Leu Val Ala Lys 1410 1415 1420
TCT TAT AGT TTG TTA TTG TCT GGG GAT AAA AAT TAT TTG ATA TCC AAT 4320 Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu He Ser Asn 1425 ' 1430 1435 1440
TTA TCT AAT ACT ATT GAG AAA ATC AAT ACT TTA GGC CTA GAT AGT AAA 4368 Leu Ser Asn Thr He Glu Lys He Asn Thr Leu Gly Leu Asp Ser Lys 1445 1450 1455
2° AAT ATA GCG TAC AAT TAC ACT GAT GAA TCT AAT AAT AAA TAT TTT GGA 4416 Asn He Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly 1460 1465 1470 GCT ATA TCT AAA ACA AGT CAA AAA AGC ATA ATA CAT TAT AAA AAA GAC 4464 Ala He Ser Lys Thr Ser Gin Lys Ser He He His Tyr Lys Lys Asp 1475 1480 1485
AGT AAA AAT ATA TTA GAA TTT TAT AAT GAC AGT ACA TTA GAA TTT AAC 4512 Ser Lys Asn He Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn 1490 1495 1500
AGT AAA GAT TTT ATT GCT GAA GAT ATA AAT GTA TTT ATG AAA GAT GAT 4560 Ser Lys Asp Phe He Ala Glu Asp He Asn Val Phe Met Lys Asp Asp 1505 1510 1515 1520
ATT AAT ACT ATA ACA GGA AAA TAC TAT GTT GAT AAT AAT ACT GAT AAA 4608
He Asn Thr He Thr Gly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys
1525 1530 1535
AGT ATA GAT TTC TCT ATT TCT TTA GTT AGT AAA AAT CAA GTA AAA GTA 4656
Ser He Asp Phe Ser He Ser Leu Val Ser Lys Asn Gin Val Lys Val 1540 1545 1550 AAT GGA TTA TAT TTA AAT GAA TCC GTA TAC TCA TCT TAC CTT GAT TTT 4704 Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe " 1555 1560 1565
GTG AAA AAT TCA GAT GGA CAC CAT AAT ACT TCT AAT TTT ATG AAT TTA 4752 Val Lys Asn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu 1570 1575 1580
TTT TTG GAC AAT ATA AGT TTC TGG AAA TTG TTT GGG TTT GAA AAT ATA 4800 Phe Leu Asp Asn He Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn He 1585 1590 1595 1600
AAT TTT GTA ATC GAT AAA TAC TTT ACC CTT GTT GGT AAA ACT AAT CTT 4848
Asn Phe Val He Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu
1605 1610 1615
GGA TAT GTA GAA TTT ATT TGT GAC AAT AAT AAA AAT ATA GAT ATA TAT 4896
Gly Tyr Val Glu Phe He Cys Asp Asn Asn Lys Asn He Asp He Tyi
1620 * 1625 1630 TTT GGT GAA TGG AAA ACA TCG TCA TCT AAA AGC ACT ATA TTT AGC GGA 4944 Phe Gly Glu Trp Lys Thr Set Ser Ser Lys Ser Thr He Pile Sei Gly 1635 1640 1645
AAT GGT AGA AAT GTT GTA GTA GAG CCT ATA TAT AAT CCT GAT ACG GGT 4992 Asn Gly Arg Asn Val Val Val Glu Pro He Tyr Asn Pro Asp Thr Gly 1650 1655 1660
GAA GAT ATA TCT ACT TCA CTA GAT TTT TCC TAT GAA CCT CTC TAT GGA 5040 Glu Asp He Ser Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly 1665 1670 1675 1680
ATA GAT AGA TAT ATA AAT AAA GTA TTG ATA GCA CCT GAT TTA TAT ACA 5088
He Asp Arg Tyr He Asn Lys Val Leu He Ala Pro Asp Leu Tyr Thr
1685 ' 1690 1695
AGT TTA ATA AAT ATT AAT ACC AAT TAT TAT TCA AAT GAG TAC TAC CCT 5136
Ser Leu He Asn He Asn Thi Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro
1700 1705 1710 GAG ATT ATA GTT CTT AAC CCA AAT ACA TTC CAC AAA AAA GTA AAT ATA 5184 Glu He He Val Leu Asn Pro Asn Thr Phe His Lys Lys Val Asn He 1715 1720 1725 AAT TTA GAT AGT TCT TCT TTT GAG TAT AAA TGG TCT ACA GAA GGA AGT 5232 Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly Ser 1730 1735 1740 GAC TTT ATT TTA GTT AGA TAC TTA GAA GAA AGT AAT AAA AAA ATA TTA 5280 Asp Phe He Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys He Leu 1745 1750 1755 ' 1760
CAA AAA ATA AGA ATC AAA GGT ATC TTA TCT AAT ACT CAA TCA TTT AAT 5328 Gin Lys He Arg He Lys Gly He Leu Ser Asn Thr Gin Ser Phe Asn
1765 1770 1775
AAA ATG AGT ATA GAT TTT AAA GAT ATT AAA AAA CTA TCA TTA GGA TAT 537G Lys Met Ser He Asp Phe Lys Asp He Lys Lys Leu Ser Leu Gly Tyr 1780 1785 1790 '
ATA ATG AGT AAT TTT AAA TCA TTT AAT TCT GAA AAT GAA TTA GAT AGA 5424 He Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg 1795 1800 1805
GAT CAT TTA GGA TTT AAA ATA ATA GAT AAT AAA ACT TAT TAC TAT GAT 5472 Asp His Leu Gly Phe Lys He He Aεp Asn Lys Thr Tyr Tyr Tyr Asp 1810 1815 1820 GAA GAT AGT AAA TTA GTT AAA GGA TTA ATC AAT ATA AAT AAT TCA TTA 5520 Glu Asp Ser Lys Leu Val Lys Gly Leu He Asn He Asn Asn Ser Leu 1825 1830 1835 1840
TTC TAT TTT GAT CCT ATA GAA TTT AAC TTA GTA ACT GGA TGG CAA ACT 5568 Phe Tyr Phe Asp Pro He Glu Phe Asn Leu Val Thr Gly Trp Gin Thr
1845 1850 1855
ATC AAT GGT AAA AAA TAT TAT TTT GAT ATA AAT ACT GGA GCA GCT TTA 5616 He Asn Gly Lys Lys Tyr Tyr Phe Asp He Asn Thr Gly Ala Ala Leu 1860 1865 1870
ACT AGT TAT AAA ATT ATT AAT GGT AAA CAC TTT TAT TTT AAT AAT GAT 5664
Thr Ser Tyr Lys He He Asn Gly Lys His Phe Tyr Phe Asn Asn Asp
1875 1880 ' 1885
GGT GTG ATG CAG TTG GGA GTA TTT AAA GGA CCT GAT GGA TTT GAA TAT 5712
Gly Val Met Gin Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
1890 1895 1900 TTT GCA CCT GCC AAT ACT CAA AAT AAT AAC ATA GAA GGT CAG GCT ATA 5760 Phe Ala Pro Ala Asn Thr Gin Asn Asn Asn He Glu Gly Gin Ala He 1905 1910 1915 ' 1920
GTT TAT CAA AGT AAA TTC TTA ACT TTG AAT GGC AAA AAA TAT TAT TTT 5808 Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe
1925 1930 ' 1935
GAT AAT AAC TCA AAA GCA GTC ACT GGA TGG AGA ATT ATT AAC AAT GAG 5856 Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg He He Asn Asn Glu 1940 1945 1950
AAA TAT TAC TTT AAT CCT AAT AAT GCT ATT GCT GCA GTC GGA TTG CAA 5904
Lys Tyr Tyr Phe Asn Pro Asn Asn Ala He Ala Ala Val Gly Leu Gin 1955 1960 1965
GTA ATT GAC AAT AAT AAG TAT TAT TTC AAT CCT GAC ACT GCT ATC ATC 5952
Val He Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala He He 1970 1975 1980 TCA AAA GGT TGG CAG ACT GTT AAT GGT AGT AGA TAC TAC TTT GAT ACT 6000 Ser Lys Gly Trp Gin Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr 1985 1990 1995 2000 GAT ACC GCT ATT GCC TTT AAT GGT TAT AAA ACT ATT GAT GGT AAA CAC 6048 Asp Thr Ala He Ala Phe Asn Gly Tyr Lys Thr He Asp Gly Lys His 2005 2010 2015
TTT TAT TTT GAT AGT GAT TGT GTA GTG AAA ATA GGT GTG TTT AGT ACC 6096 Phe Tyr Phe Asp Ser Asp Cys Val Val Lys He Gly Val Phe Ser Thr 2020 2025 " 2030
TCT AAT GGA TTT GAA TAT TTT GCA CCT GCT AAT ACT TAT AAT AAT AAC 6144 Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn 2035 2040 2045
ATA GAA GGT CAG GCT ATA GTT TAT CAA AGT AAA TTC TTA ACT TTG AAT 6192 He GJu Gly Gin Ala He Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn 2050 2055 2060
GGT AAA AAA TAT TAC TTT GAT AAT AAC TCA AAA GCA GTT ACC GGA TTG 6240 Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly Leu 2065 2070 2075 2080
CAA ACT ATT GAT AGT AAA AAA TAT TAC TTT AAT ACT AAC ACT GCT GAA 6288 Gin Thr He Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu 2085 2090 2095 GCA GCT ACT GGA TGG CAA ACT ATT GAT GGT AAA AAA TAT TAC TTT AAT 6336 Ala Ala Thr Gly Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn 2100 2105 ' 21.10
ACT AAC ACT GCT GAA GCA GCT ACT GGA TGG CAA ACT ATT GAT GGT AAA 6384 Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr He Asp Gly Lys 2115 2120 2125
AAA TAT TAC TTT AAT ACT AAC ACT GCT ATA GCT TCA ACT GGT TAT ACA 6432 Lys Tyr Tyr Phe Asn Thr Asn Thr Ala He Ala Ser Thr Gly Tyr Thr 2130 ' 2135 2140
ATT ATT AAT GGT AAA CAT TTT TAT TTT AAT ACT GAT GGT ATT ATG CAG 6480 He He Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly He Met Gin 2145 2150 2155 2160
ATA GGA GTG TTT AAA GGA CCT AAT GGA TTT GAA TAT TTT GCA CCT GCT 6528 He Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala 2165 2170 2175 AAT ACG GAT GCT AAC AAC ATA GAA GGT CAA GCT ATA CTT TAC CAA AAT 6576 Asn Thr Asp Ala Asn Aεn He Glu Gly Gin Ala He Leu Tyr Gin Asn 2180 2185 2190
GAA TTC TTA ACT TTG AAT GGT AAA AAA TAT TAC TTT GGT AGT GAC TCA 6624 Glu Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205
AAA GCA GTT ACT GGA TGG AGA ATT ATT AAC AAT AAG AAA TAT TAC TTT 6672 Lys Ala Val Thr Gly Trp Arg He He Asn Asn Lys Lys Tyr Tyr Phe 2210 2215 2220
AAT CCT AAT AAT GCT ATT GCT GCA ATT CAT CTA TGC ACT ATA AAT AAT 6720
Asn Pio Asn Asn Ala He Ala Ala He His Leu Cys Thr He Asn Asn 2225 2230 2235 2240
GAC AAG TAT TAC TTT AGT TAT GAT GGA ATT CTT CAA AAT GGA TAT ATT 6768
Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly He Leu Gin Asn Gly Tyr He 2245 2250 2255 ACT ATT GAA AGA AAT AAT TTC TAT TTT GAT GCT AAT AAT GAA TCT AAA 6816 Thr He Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys 2260 2265 2270 ATG GTA ACA GGA GTA TTT AAA GGA CCT AAT GGA TTT GAG TAT TTT GCA 6864 Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala 2275 2280 2285 CCT GCT AAT ACT CAC AAT AAT AAC ATA GAA GGT CAG GCT ATA GTT TAC 6912 Pro Ala Asn Thr His Asn Asn Asn He Glu Gly Gin Ala He Val Tyr 2290 2295 2300
CAG AAC AAA TTC TTA ACT TTG AAT GGC AAA AAA TAT TAT TTT GAT AAT 6960 Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn 2305 2310 2315 2320
GAC TCA AAA GCA GTT ACT GGA TGG CAA ACC ATT GAT GGT AAA AAA TAT 7008 Asp Ser Lys Ala Val Thr Gly Trp Gin Thr He Asp Gly Lys Lys Tyr ' 2325 2330 * 2335
TAC TTT AAT CTT AAC ACT GCT GAA GCA GCT ACT GGA TGG CAA ACT ATT 7056
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr He 2340 2345 2350
GAT GGT AAA AAA TAT TAC TTT AAT CTT AAC ACT GCT GAA GCA GCT ACT 7104
Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr 2355 2360 2365 GGA TGG CAA ACT ATT GAT GGT AAA AAA TAT TAC TTT AAT ACT AAC ACT 7152 Gly Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 2370 2375 ' 2380
TTC ATA GCC TCA ACT GGT TAT ACA AGT ATT AAT GGT AAA CAT TTT TAT 7200 phe He Ala Ser Thr Gly Tyr Thr Ser He Asn Gly Lys His Phe Tyr 2385 2390 2395 2400
TTT AAT ACT GAT GGT ATT ATG CAG ATA GGA GTG TTT AAA GGA CCT AAT 7248 Phe Asn Thr Asp Gly He Met Gin He Gly Val Phe Lys Gly Pro Asn 2405 2410 2415
GGA TTT GAA TAC TTT GCA CCT GCT AAT ACG GAT GCT AAC AAC ATA GAA 7296 Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu 2420 2425 2430
GGT CAA GCT ATA CTT TAC CAA AAT AAA TTC TTA ACT TTG AAT GGT AAA 7344 Gly Gin Ala He Leu Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445 AAA TAT TAC TTT GGT AGT GAC TCA AAA GCA GTT ACC GGA CTG CGA ACT 7392 Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr 2450 2455 2460
ATT GAT GGT AAA AAA TAT TAC TTT AAT ACT AAC ACT GCT GTT GCA GTT 7440 He Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val 2465 ' 2470 ' 2475 2480
ACT GGA TGG CAA ACT ATT AAT GGT AAA AAA TAC TAC TTT AAT ACT AAC 7488 Thr Gly Trp Gin Thr He Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 2485 2490 2495
ACT TCT ATA GCT TCA ACT GGT TAT ACA ATT ATT AGT GGT AAA CAT TTT 7536
Thr Ser He Ala Ser Thr Gly Tyr Thr He He Ser Gly Lys His Phe 2500 2505 2510
TAT TTT AAT ACT GAT GGT ATT ATG CAG ATA GGA GTG TTT AAA GGA CCT 7584
Tyr Phe Asn Thr Asp Gly He Met Gin He Gly Val Phe Lys Gly Pro 2515 2520 2525 GAT GGA TTT GAA TAC TTT GCA CCT GCT AAT ACA GAT GCT AAC AAT ATA 7632 Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He 2530 2535 2540 GAA GGT CAA GCT ATA CGT TAT CAA AAT AGA TTC CTA TAT TTA CAT GAC 7680 Glu Gly Gin Ala He Arg Tyr Gin Asn Arg Phe Leu Tyr Leu His Asp 2545 2550 2555 2560 AAT ATA TAT TAT TTT GGT AAT AAT TCA AAA GCG GCT ACT GGT TGG GTA 7728 Asn He Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val 2565 2570 2575
ACT ATT GAT GGT AAT AGA TAT TAC TTC GAG CCT AAT ACA GCT ATG GGT 7776 Thr He Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly
2580 2585 2590
GCG AAT GGT TAT AAA ACT ATT GAT AAT AAA AAT TTT TAC TTT AGA AAT 7824 Ala Asn Gly Tyr Lys Thr He Asp Asn Lys Asn Phe Tyr Phe Arg Asn 2595 2600 2605
GGT TTA CCT CAG ATA GGA GTG TTT AAA GGG TCT AAT GGA TTT GAA TAC 7872 Gly Leu Pro Gin He Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr 2610 2615 2620
TTT GCA CCT GCT AAT ACG GAT GCT AAC AAT ATA GAA GGT CAA GCT ATA 7920 Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu Gly Gin Ala He 2625 2630 2635 2640 CGT TAT CAA AAT AGA TTC CTA CAT TTA CTT GGA AAA ATA TAT TAC TTT 7968 Arg Tyi Gin Asn Arg Phe Leu His Leu Leu Gly Lys He Tyr Tyr Phe 2645 2650 ' 2655
GGT AAT AAT TCA AAA GCA GTT ACT GGA TGG CAA ACT ATT AAT GGT AAA 8016 Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gin Thr He Asn Gly Lys
2660 ' 2665 2670
GTA TAT TAC TTT ATG CCT GAT ACT GCT ATG GCT GCA GCT GGT GGA CTT 8064 Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680 2685
TTC GAG ATT GAT GGT GTT ATA TAT TTC TTT GGT GTT GAT GGA GTA AAA 8112 Phe Glu He Asp Gly Val He Tyr Phe Phe Gly Val Asp Gly Val Lys 2690 2695 2700
GCC CCT GGG ATA TAT GGC TAA 8133
Ala Pro Gly He Tyr Gly 2705 2710 (2) INFORMATION FOR SEQ ID NO : 6 :
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2710 ammo acids
Figure imgf000232_0001
(D) TOPOLOGY, linear
(n) MOLECULE TYPE: protein
(κi) SEQUENCE DESCRIPTION: SEQ ID NO : 6.
Met Ser Leu He Ser Lys Glu Glu Leu He Lys Leu Ala Tyr Ser He 1 5 10 15
Arg Pro Arg Glu Asn Glu Tyr Lys Thr He Leu Thr Asn Leu Asp Glu 20 25 30
Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gin Leu 5 40 45 Lys Lys Leu Asn Glu Ser He Asp Val Phe Met Asn Lys Tyr Lys Thr 50 55 60
Ser Sei Arg Asn Arg Ala Leu Ser Acn Leu Lys Lys Asp He Leu Lys 65 70 75 80 Glu Val He Leu He Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn 85 90 95
Leu His Phe Val Trp He Gly Gly Glu Val Ser Asp He Ala Leu Glu 100 ' 105 110
Tyr He Lys Gin Trp Ala Asp He Asn Ala Glu Tyr Asn He Lys Leu 115 120 125 Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala He 130 135 140
Val Glu Ser Ser Thr Thr Glu Ala Leu Gin Leu Leu Glu Glu Glu He 145 150 155 160
Gin Asn Pro Gin Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu
165 170 ' 175
Phe He Tyr Asp Arg Gin Lys Arg Phe He Asn Tyr Tyr Lys Ser Gin 180 185 190
He Asn Lys Pro Thr Val Pro Thr He Asp Asp He He Lys Ser His 195 200 205 Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg
210 215 220
Thr Asn Ser Leu Arg Lys He Asn Ser Asn His Glv He Asn He Arg
225 230 235 240
Ala Asn Ser Leu Phe Thr Glu Gin Glu Leu Leu Asn He Tyr Ser Gin 245 250 255
Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp He Val Ai g 260 265 270
Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met
275 280 2B5 Leu Pro Gly He His Ser Asp Leu Phe Lys Thr He Ser Arg Pro Ser
290 295 300
Ser He Gly Leu Asp Arg Trp Glu Met He Lys Leu Glu Ala He Met 305 310 315 320
Lys Tyr Lys Lys Tyr He Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys 325 330 335
Leu Asp Gin Gin Leu Lys Asp Asn Phe Lys Leu He He Glu Ser Lys 340 ' 345 350
Ser Glu Lys Ser Glu He Phe Ser Lys Leu Glu Asn Leu Asn Val Ser 355 360 365 Asp Leu Glu He Lys He Ala Phe Ala Leu Gly Ser Val He Asn Gin 370 375 380
Ala Leu He Ser Lys Gin Gly Ser Tyr Leu Thr Asn Leu Val He Glu
385 390 395 400
Gin Val Lys Asn Arg Tyr Gin Phe Leu Asn Gin His Leu Asn Pro Ala
405 410 15
He Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys He Phe His Asp 420 425 430
Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys
435 440 445 He Ala Pro Tyr Leu Gin Val Gly Phe Met Pro Glu Ala Arg Ser Thr 450 455 460
He Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe 465 470 475 480
He Asn Leu Gin Glu Asn Thr He Glu Lys Thr Leu Lys Ala Ser Asp 485 490 495 Leu He Glu Phe Lys Phe Pro GLu Asn Asn Leu Ser Gin Leu Thr Glu
500 505 510
Gin Glu He Asn Ser Leu Trp Ser Phe Asp Gin Ala Ser Ala Lys Tyr 515 520 525
Gin Phe Glu Lys Tyr Val Arg Asp yr Thr Gly Gly Ser Leu Ser Glu 530 535 540
Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr 545 550 555 560
Leu Leu Asn Asn Lys He Pro Ser Asn Asn Val Glu Glu Ala Gly Ser 565 570 575 Lvs Asn Tyr Val His Tyr He He Gin Leu Gin Gly Asp Asp He Ser
580 585 590
'lyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser He 595 " 600 605
He He Gin Arg Asn Met Asn Glu Ser Ala Lys Ser ryr Phe Leu Ser 610 615 620
Asp Asp Gly Glu Ser He Leu Glu Leu Asn Lys Tyr Arg He Pro Glu 625 630 635 640
Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe He Gly His Gly 645 ' 650 655 Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser
660 665 670
Aεn Glu He Ser Ser Phe Leu Asp Thr He Lys Leu ASD He
675 680 685
Pro Lvs Asn Va Glu Val Asn Leu Leu Glv Cys Asn M^t Phe Ser 690 695 700
Figure imgf000234_0001
He Met Asp Lys He Thr Ser Thr Leu Pro Asp Val Aεn Lys Asn Ser
725 730 735 He Thr He Glv Ala Asn Gin Tyr Glu Val Arg He Asn Ser Glu Gly
740 745 750
Arg Lys Glu Leu Leu Ala His Ser Gly Lyε Trp He Asn Lys Glu Glu
755 760 * 65
Ala He Met Ser Asp Leu Ser Ser Lys Glu Tyr He Phe Phe Asp Ser
770 775 ' 780
He Aεp Asn Lys Leu Lys Ala Lys Ser Lys Asn He Pro Gly Leu Ala ^85 790 795 800
Ser He Ser Glu Asp He Lys Thr Leu Leu Leu Aεp Ala Ser Val Ser
805 810 815 Pro Asp Thr Lys Phe He Leu Asn Asn Leu Lys Leu Asn He Glu Ser 820 825 830
Ser He Gly Asp Tyr He Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn 835 840 845
He He His Asn Ser He Asp Asp Leu He Asp Glu Phe Asn Leu Leu 850 855 860
Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu 865 870 ' 875 880
Asp Glu Lys Tyr Leu He Ser Phe Glu Asp He Ser Lys Asn Asn Ser 885 890 ' 895
Thr Tyr Ser Val Arg Phe He Asn Lys Ser Asn Gly Glu Ser Val Tyr 900 905 910
Val Glu Thr Glu Lys Glu He Phe Ser Lys Tyr Ser Glu His He Thr 915 920 925
Lys Glu He Ser Thr He Lys Asn Ser He He Thr Asp Val Asn Gly 930 935 940
Asn Leu Leu Asp Asn He Gin Leu Asp His Thr Ser Gin Val Asn Thr 945 950 955 960
Leu Asn Ala Ala Phe Phe He Gin Ser Leu He Aεp Tyr Ser Ser Asn 965 970 975
Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gin Leu Tyr 980 985 990
Ala Gin Leu Phe Ser Thr Gly Leu Asn Thr He Tyr Asp Ser He Gin 995 1000 ' 1005
Leu Val Asn Leu He Ser Asn Ala Val Asn Asp Thr He Asn Val Leu 1010 1015 1020 Pro Thr He Thr Glu Gly He Pro He Val Ser Thr He Leu Asp Gly 1025 1030 1035 1040
He Asn Leu Gly Ala Ala He Lys Glu Leu Leu Asp Glu His Asp Pro 1045 1050 1055
Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val Leu Ala He Asn 1060 1065 1070
Met Ser Leu Ser He Ala Ala Thr Val Ala Ser He Val Gly He Gly 1075 1080 1085
Ala Glu Val Thr He Phe Leu Leu Pro He Ala Gly He Ser Ala Gly 1090 1095 1100 He Pro Ser Leu Val Asn Asn Glu Leu He Leu His Asp Lys Ala Thr 1105 1110 1115 1120
Ser Val Val Asn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly 1125 1130 ' 1135
Pro Leu Lys Thr Glu Asp Asp Lys He Leu Val Pro He Asp Asp Leu 1140 1145 1150
Val He Ser Glu He Asp Phe Asn Asn Asn Ser He Lys Leu Gly Thr 1155 1160 1165
Cys Asn He Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly 1170 1175 1180 Asn He Asp His Phe Phe Ser Ser Pro Ser He Ser Ser His He Pro 1185 1190 1195 1200
Ser Leu Ser He Tyr Ser Ala He Gly He Glu Thr Glu Asn Leu Asp 1205 1210 1215
Phe Ser Lys Lys He Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe 1220 1225 1230 Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp 1235 1240 1245
Gly Thr Arg Leu Leu Asp Ser He Arg Asp Leu Tyr Pro Gly Lys Phe 1250 1255 1260
Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala He Thi Thr Leu Lys 1265 * 1270 1275 1280
Pro Val Tyr Glu Asp Thr Asn He Lys He Lys Leu Asp Lys Asp Thr ' 1285 1290 ' ' 1295
Arg Asn Phe He Met Pro Thr He Thr Thr Asn Glu He Arg Asn Lys 1300 1305 1310
Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Sei Leu Leu Leu 1315 1320 1325
Ser Ser Tyr Pro He Ser Thr Asn He Asn Leu Ser Lys Asp Asp Leu 1330 1335 1340
Trp He Phe Asn lie Asp Asn Glu Val Arg Glu He Ser He Glu Asn 1345 1350 1355 1360
Gly Thr He Lys Lys Gly Lvs Leu He Lys Asp Val Leu Ser Lys He ' ' 1365 ' 1370 1375
Asp He Asn Lys Asn Lys Leu He He Gly Asn Gin Thr He Asp Phe 1380 1385 1390 Ser Gly Asp He Asp Asn Lys Asp Arg Tyr He Phe Leu Thr Cys Glu 1395 1400 1405
Leu Asp Asp Lys He Ser Leu He He Glu He Asn Leu Val Ala Lys 1410 1415 1420
Sei Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu lie Ser Asn 1425 ' 1430 1435 1440
Leu Ser Asn Thr He Glu Lys He Asn Thr Leu Gly Leu Asp Ser Lys 1445 ' 1450 1455
Asn He Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly 1460 1465 1470 Ala He Ser Lys Thr Ser Gin Lys Ser He He His Tyr Lys Lys Asp
1475 1480 1485
Ser Lys Asn He Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn 1490 1495 1500
Ser Lys Asp Phe He Ala Glu Asp He Asn Val Phe Met Lys Asp Asp 1505 ' 1510 1515 1520
He Asn Thr He Thr Gly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys 1525 1530 1535
Ser He Asp Phe Ser He Ser Leu Val Ser Lys Asn Gin Val Lys Val 1540 1545 1550 Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe 1555 1560 1565
Val Lys Asn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu 1570 1575 1580
Phe Leu Asp Asn He Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn He 1585 1590 1595 1600 Asn Phe Val He Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu
1605 1610 1615
Gly Tyr Val Glu Phe He Cys Asp Asn Asn Lys Asn He Asp He Tyr 1620 1625 1630
Phe Gly Glu Trp Lys Thr Ser Ser Ser Lys Ser Thr He Phe Ser Gly 1635 1640 1645
Asn Gly Arg Asn Val Val Val Glu Pro He Tyr Asn Pro Asp Thr Gly 1650 1655 1660
Glu Asp He Ser Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly 1665 1670 1675 ' 1680 He Asp Arg Tyr He Asn Lyε Val Leu He Ala Pro Asp Leu Tyr Thr
1685 1690 1695
Ser Leu He Asn He Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro 1700 1705 1710
Glu He He Val Leu Asn Pro Asn Thr Phe His Lyr Lys Val Asn He 1715 1720 1725
Aεn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly Ser 1730 1735 1740
Asp Phe He Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys He Leu 1745 1750 1755 * 1760 Gin Lys He Arg He Lys Gly He Leu Ser Asn Thr Gin Ser Phe Asn
1765 1770 1775
Lys Met Ser He Asp Phe Lys Asp He Lys Lys Leu Ser Leu Gly Tyr 1780 1785 1790
He Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg 1795 * 1800 1805
Asp His Leu Gly Phe Lys He He Asp Asn Lys Thr Tyr Tyr Tyr Asp 1810 1815 1820
Glu Asp Ser Lys Leu Val Lys Gly Leu He Asn He Asn Asn Ser Leu 1825 1830 1835 1840 Phe Tyr Phe Asp Pro He Glu Phe Asn Leu Val Thr Gly Trp Gin Thr
1845 1850 1855
He Asn Gly Lys Lys Tyr Tyr Phe Asp He Asn Thr Gly Ala Ala Leu 1860 ' 1865 1870
Thr Ser Tyr Lys He He Asn Gly Lys His Phe Tyr Phe Asn Asn Asp
1875 1880 1885
Gly Val Met Gin Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr 1890 1895 1900
Phe Ala Pro Ala Asn Thr Gin Asn Asn Asn He Glu Gly Gin Ala He
1905 1910 1915 1920 Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe 1925 1930 * 1935
Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg He He Asn Asn Glu 1940 1945 1950
Lys Tyr Tyr Phe Asn Pro Asn Asn Ala He Ala Ala Val Gly Leu Gin 1955 1960 1965
Val He Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala He He 1970 1975 1980
Ser Lys Gl Trp Gin Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr 1985 1990 1995 2000
Asp Thr Ala He Ala Phe Asn Gly Tyr Lys Thr He Asp Gly Lys Hiε 2005 2010 2015
Phe Tyr Phe Asp Ser Asp Cys Val Val Lys He Gly Val Phe Ser Thr 2020 2025 2030
Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Aεn Asn Asn 2035 2040 2045
He Glu Gly Gin Ala He Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn 2050 2055 2060
Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly Leu 2065 ' 2070 2075 2080
Gin Thr He Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu 2085 2090 2095
Ala Ala Thr Glv Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn 100 2105 2110
Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr He Asp Gly Lys 2115 2120 2125
Lys Tyr Tyr Phe Asn Thr Asn Thr Ala He Ala Ser Thr Gly Tyr Thr
2130 2135 2140
He He Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly He Met Gin 2145 2150 ' 2155 2160
He Gly Val Phe Lvs Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala 2165 2170 2175
Asn Thr Asp Ala Asn Asn He Glu Gly Gin Ala He Leu Tyr Gin Asn 2180 2185 2190
Glu Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser 2195 2200 2205 Lys Ala Val Thr Gly Trp Arg He He Asn Asn Lys Lys Tyr Tyr Phe
2210 2215 2220
Asn Pro Asn Asn Ala He Ala Ala He His Leu Cys Thr He Asn Asn 2225 2230 2235 2240
Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly He Leu Gin Asn Gly Tyr He 2245 2250 2255
Thr He Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys 2260 2265 2270
Met Val Thr Gly Val Phe Lvs Gly Pro Asn Gly Phe Glu Tyr Phe Ala 2275 * ' 2280 2285 Pro Ala Asn Thr His Asn Asn Asn He Glu Gly Gin Ala He Val Tyr
2290 2295 2300
Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn 5 2305 2310 2315 2320
Asp Ser Lys Ala Val Thr Gly Trp Gin Thr He Asp Gly Lys Lys Tyr 2325 2330 2335
10 Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr He
2340 2345 2350
Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr 2355 2360 2365
15
Gly Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 2370 2375 " 2380
Phe He Ala Ser Thr Gly Tyr Thr Ser He Asn Gly Lys His Phe Tyr 20 2385 2390 2395 2400
Phe Asn Thr Asp Gly He Met Gin He Gly Val Phe Lys Gly Pro Asn 2405 2410 2415
25 Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu
2420 2425 2430
Gly Gin Ala He Leu Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys 2435 2440 2445
30
Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr 2450 2455 2460
He Aε p Gly Lys Ly∑ Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val i:> 2465 2470 2475 2480
Thr Gly Trp Gin Thr He Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn 2485 2490 2495
40 Thr Ser He Ala Ser Thr Gly Tyr Thr He He Ser Gly Lys His Phe
2500 2505 2510
Tyr Phe Asn Thr Asp Gly He Met Gin He Gly Val Phe Lys Gly Pro 2515 2520 2525
45
Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He 2530 ' 2535 2540
Glu Gly Gin Ala He Arg Tyr Gin Asn Arg Phe Leu Tyr Leu His Asp 50 2545 ' 2550 2555 2560
Asn He Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val 2565 2570 2575
55 Thr He Aεp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly
2580 2585 2590
Ala Asn Gly Tyr Lys Thr He Asp Asn Lys Asn Phe Tyr Phe Arg Asn 2595 2600 2605
60
Gly Leu Pro Gin He Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr 2610 2615 2620
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu Gly Gin Ala He 65 2625 2630 2635 2640
Arg Tyr Gin Asn Arg Phe Leu His Leu Leu Gly Lys He Tyr Tyr Phe 2645 2650 2655 Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gin Thr He Asn Gly Lys 2660 2665 2670
Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu 2675 2680 2685
Phe Glu He Asp Gly Val He Tyr Phe Phe Gly Val Asp Gly Val Lys 2690 2695 2700 Ala Pro Gly He Tyr Gly 2705 2710
(2) INFORMATION FOR SEQ ID NO : : (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 811 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protem
(Xl ) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : Ser Tyi Lys He He Asn Gly Lys His Phe Tyr Phe Asn Asn Asp Gly
1 5 10 15
Val Met Gin Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe 20 25 30
Ala Pro Ala Asn Thr Gin Asn Asn Asn He Glu Gly Gin Ala He Val
35 40 45
Tyr Gin Ser Lys Phe Leu Thr Leu Asn Gly Lys Lyε Tyr Tyr Phe Asp 50 55 60
Asn Asn Ser Lys Ala Val Thr Gly Trp Arg He He Asn Asn Glu Lyε 65 70 75 80
Tyr Tyr Phe Asn Pro Asn Asn Ala He Ala Ala Val Gly Leu Gin Val 85 90 95
He Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala He He Ser 100 105 110
Lvs Gly Trp Gin Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr Asp 115 120 ' 125
Thr Ala He Ala Phe Asn Gly Tyr Lys Thr He Asp Gly Lys His Phe 130 135 140
Tyr Phe Asp Ser Asp Cys Val Val Lys He Gly Val Phe Ser Thr Ser
145 150 155 160
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn He
165 170 175
Glu Gly Gin Ala He Val Tyr Gin Ser Lys Phe Leu Thr Leu Asn Gly
180 185 190
Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly Leu Gin
195 ' 200 205
Thr He Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu Ala 210 ' 215 220
Ala Thr Gly Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn Thr
225 230 235 ' ' 240 Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr He Asp Gly Lys Lys 245 250 255
Tyr Tyr Phe Asn Thr Asn Thr Ala He Ala Ser Thr Gly Tyr Thr He 260 265 270
He Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly He Met Gin He 275 280 285 Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
290 295 300
Thr Asp Ala Asn Aεn He Glu Gly Gin Ala He Leu Tyr Gin Asn Glu 305 310 315 320
Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys 325 ' ' 330 335
Ala Val Thr Gly Trp Arg He He Asn Asn Lys Lys Tyr Tyr Phe Asn 340 345 ' 350
Pro Asn Asn Ala He Ala Ala He His Leu Cys Thr He Asn Asn Asp 355 360 ' 365 Lys Tyr Tyr Phe Ser Tyr Asp Gly He Leu Gin Asn Gly Tyr He Thr
370 375 380
He Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met 385 390 395 400
Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro 405 ' 410 415
Ala Asn Thr His Asn Asn Asn He Glu Gly Gin Ala He Val Tvr Gin
420 425 430
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lyε Tyr Tyr Phe Aεp Aεn Asp 435 440 ' 445
Ser Lyε Ala Val Thr Gly Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr 450 455 460
Pl:e Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gin Thr He Asp
465 470 475 480
Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Tnr Gly
485 490 495
Trp Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe 500 505 " 510
He Ala Ser Thr Gly Tyr Thr Ser He Asn Gly Lyε His Phe Tyr Phe
515 520 525 Asn Thr Asp Gly He Met Gin He Gly Val Phe Lys Gly Pro Asn Gly
530 535 540
Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu Gly
545 550 555 560
Gin Ala He Leu Tyr Gin Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys
565 ' 570 575
Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr He 580 585 590
Asp Gly Lys Lyε Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr 595 600 605 Gly Trp Gin Thr He Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr 610 615 620
Ser He Ala Ser Thr Gly Tyr Thr He He Ser Gly Lyε His Phe Tyr
625 630 635 640
Phe Asn Thr Asp Gly He Met Gin He Gly Val Phe Lys Gly Pro Asp
645 650 ' 655
Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu
660 665 670
Gly Gin Ala He Arg Tyr Gin Asn Arg Phe Leu Tyr Leu His Asp Asn
675 680 ' 685
He Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr 690 695 700
He Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala
705 710 715 720
Asn Gly Tyr Lys Thr He Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly 725 " 730 735
Leu Pro Gin He Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe 740 745 750
Ala Pro Ala Asn Thr Asp Ala Asn Asn He Glu Gly Gin Ala He Arg 755 760 ' 765
Tyr Gin Asn Arg Phe Leu His Leu Leu Gly Lys He Tyr Tyr Phe Gly
770 775 " 780
Asn Asn Ser Lys Ala Val Thr Gly Trp Gin Thr He Asn Gly Lys Val 785 790 ' 795 ' 800 lyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala 805 810 NFORMATION FOR SEQ ID NO : 8 : d) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 91 ammo acids
(B) TYPE: ammo acid (C) STRANDEDNESS. unknown
(D) TOPOLOGY: unknown
'ii) MOLECULE TYPE: protein (κi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Ser Tyr Lys He He Asn Gly Lys His Phe Tyr Phe Aεn Asn Asp Gly 1 5 10 15 Val Met Gin Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe
20 25 30
Ala Pro Ala Asn Thr Gin Asn Asn Asn He Glu Gly Gin Ala He Val 35 40 45
Tyr TJln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp 50 55 ' 60
Asn Asn Ser Lys Ala Val Thr Gly Trp Arg He Ho Asn Asn Glu Lys 65 70 75 80
Tyr Tyr Phe Asn Pro Asn Asn Ala He Ala Ala
85 90 (2) INFORMATION FOR SEQ ID NO: 9:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7101 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ul MOLECULE TYPE: DNA (genomic)
(IX) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..7098
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :
ATG AGT TTA GTT AAT AGA AAA CAG TTA GAA AAA ATG GCA AAT GTA AGA 48
Met Ser Leu Val Asn Arg Lys Gin Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15
TTT CGT ACT CAA GAA GAT GAA TAT GTT GCA ATA TTG GAT GCT TTA GAA 96 Phe Arg Thr Gin Glu Asp Glu Tyr Val Ala He Leu Asp Ala Leu Glu 20 25 30 GAA TAT CAT AAT ATG TCA GAG AAT ACT GTA GTC GAA AAA TAT TTA AAA 144 Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys
Figure imgf000243_0001
TTA AAA GAT ATA AAT AGT TTA ACA GAT ATT TAT ATA GAT ACA TAT AAA 192 Leu Lys Asp He Asn Ser Leu Thr Asp He Tyr He Asp Thr Tyr Lys 50 55 60
AAA TCT GGT AGA AAT AAA GCC TTA AAA AAA TTT AAG GAA TAT CTA GTT 240 Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val 65 70 75 80
ACA GAA GTA TTA GAG CTA AAG AAT AAT AAT TTA ACT CCA GTT GAG AAA 288
Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys
85 ' 90 95
AAT TTA CAT TTT GTT TGG ATT GGA GGT CAA ATA AAT GAC ACT GCT ATT 336
Asn Leu His Phe Val Trp He Gly Gly Gin He Asn Asp Thr Ala He
100 105 110 AAT TAT ATA AAT CAA TGG AAA GAT GTA AAT AGT GAT TAT AAT GTT AAT 384 Asn Tyr He Asn Gin Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn 115 ' 120 125
GTT TTT TAT GAT AGT AAT GCA TTT TTG ATA AAC ACA TTG AAA AAA ACT 432 Val Phe Tyr Asp Ser Asn Ala Phe Leu He Asn Thr Leu Lys Lys Thr 130 ' 135 140
GTA GTA GAA TCA GCA ATA AAT GAT ACA CTT GAA TCA TTT AGA GAA AAC 480 Val Val Glu Ser Ala He Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160
TTA AAT GAC CCT AGA TTT GAC TAT AAT AAA TTC TTC AGA AAA CGT ATG 528 Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met 165 170 175
GAA ATA ATT TAT GAT AAA CAG AAA AAT TTC ATA AAC TAC TAT AAA GCT 576 Glu He He Tyr Asp Lys Gin Lys Asn Phe He Asn Tyr Tyr Lys Ala 180 185 190 CAA AGA GAA GAA AAT CCT GAA CTT ATA ATT GAT GAT ATT GTA AAG ACA 624 Gin Arg Glu Glu Asn Pro Glu Leu He He Asp Asp He Val Lys Thr 195 200 205 TAT CTT TCA AAT GAG TAT TCA AAG GAG ATA GAT GAA CTT AAT ACC TAT 672 Tyr Leu Ser Asn Glu Tyr Ser Lys Glu He Asp Glu Leu Asn Thr Tyr 210 215 220
ATT GAA GAA TCC TTA AAT AAA ATT ACA CAG AAT AGT GGA AAT GAT GTT 720 He Glu Glu Ser Leu Asn Lys He Thr Gin Asn Ser Gly Asn Asp Val 225 230 235 240
AGA AAC TTT GAA GAA TTT AAA AAT GGA GAG TCA TTC AAC TTA TAT GAA 768 Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu 245 250 255
CAA GAG TTG GTA GAA AGG TGG AAT TTA GCT GCT GCT TCT GAC ATA TTA 816 Gin Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Aεp He Leu 260 265 270
AGA ATA TCT GCA TTA AAA GAA ATT GGT GGT ATG TAT TTA GAT GTT GAT 864 Arg He Ser Ala Leu Lys Glu He Gly Gly Met Tyr Leu Asp Val Asp 275 280 285
ATG TTA CCA GGA ATA CAA CCA GAC TTA TTT GAG TCT ATA GAG AAA CCT 912 Met Leu Pro Gly He Gin Pro Asp Leu Phe Glu Ser He Glu Lys Pro 290 295 300 AGT TCA GTA ACA GTG GAT TTT TGG GAA ATG ACA AAG TTA GAA GCT ATA 960 Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala He 105 310 315 ' 320
ATG AAA TAC AAA GAA TAT ATA CCA GAA TAT ACC TCA GAA CAT TTT GAC 1008 Met Lys Tyr Lys Glu Tyr He Pro Glu Tyr Thr Ser Glu His Phe Asp
325 330 335
ATG TTA GAC GAA GAA GTT CAA AGT AGT TTT GAA TCT GTT CTA GCT TCT 1056 Met Leu Asp Glu Glu Val Gin Ser Ser Phe Glu Ser Val Leu Ala Ser 340 345 350
AAG TCA GAT AAA TCA GAA ATA TTC TCA TCA CTT GGT GAT ATG GAG GCA 1104
Lys Ser Asp Lys Ser Glu He Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365
TCA CCA CTA GAA GTT AAA ATT GCA TTT AAT AGT AAG GGT ATT ATA AAT 1152
Ser Pro Leu Glu Val Lys He Ala Phe Asn Ser Lys Gly He He Asn
370 375 380 CAA GGG CTA ATT TCT GTG AAA GAC TCA TAT TGT AGC AAT TTA ATA GTA 1200 Gin Gly Leu He Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu He Val 385 ' 390 395 400
AAA CAA ATC GAG AAT AGA TAT AAA ATA TTG AAT AAT AGT TTA AAT CCA 1248 Lys Gin He Glu Asn Arg Tyr Lys He Leu Asn Asn Ser Leu Asn Pro
405 410 415
GCT ATT AGC GAG GAT AAT GAT TTT AAT ACT ACA ACG AAT ACC TTT ATT 1296 Ala He Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe He 420 425 430
GAT AGT ATA ATG GCT GAA GCT AAT GCA GAT AAT GGT AGA TTT ATG ATG 1344 Asp Ser He Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445
GAA CTA GGA AAG TAT TTA AGA GTT GGT TTC TTC CCA GAT GTT AAA ACT 1392 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 ' 455 460 ACT ATT AAC TTA AGT GGC CCT GAA GCA TAT GCG GCA GCT TAT CAA GAT 1440 Thr He Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gin Asp 465 470 475 480 TTA TTA ATG TTT AAA GAA GGC AGT ATG AAT ATC CAT TTG ATA GAA GCT 1488
Leu Leu Met Phe Lys Glu Gly Ser Met Asn He His Leu He Glu Ala 485 490 495 GAT TTA AGA AAC TTT GAA ATC TCT AAA ACT AAT ATT TCT CAA TCA ACT 1536
Asp Leu Arg Asn Phe Glu He Ser Lys Thr Asn He Ser Gin Ser Thr 500 505 510
GAA CAA GAA ATG GCT AGC TTA TGG TCA TTT GAC GAT GCA AGA GCT AAA 1 84 Glu Gin Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys 515 520 525
GCT CAA TTT GAA GAA TAT AAA AGG AAT TAT TTT GAA GGT TCT CTT GGT 1632
Ala Gin Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540
GAA GAT GAT AAT CTT GAT TTT TCT CAA AAT ATA GTA GTT GAC AAG GAG 1680
Glu Asp Asp Asn Leu Asp Phe Ser Gin Asn He Val Val Asp Lys Glu
545 550 555 560
TAT CTT TTA GAA AAA ATA TCT TCA TTA GCA AGA AGT TCA GAG AGA GGA 1728
Tyr Leu Leu Glu Lys He Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 ' 575 TAT ATA CAC TAT ATT GTT CAG TTA CAA GGA GAT AAA ATT AGT TAT GAA 1776
Tyr He His Tyr He Val Gin Leu Gin Gly Asp Lys He Ser Tyr Glu 580 585 590
GCA GCA TGT AAC TTA TTT GCA AAG ACT CCT TAT GAT AGT GTA CTG TTT 1824 Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605
CAG AAA AAT ATA GAA GAT TCA GAA ATT GCA TAT TAT TAT AAT CCT GGA 1872
Gin Lys Asn He Glu Asp Ser Glu He Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620
GAT GGT GAA ATA CAA GAA ATA GAC AAG TAT AAA ATT CCA AGT ATA ATT 1920
Asp Gly Glu He Gin Glu He Asp Lys Tyr Lys He Pro Ser He He
625 630 635 640
TCT GAT AGA CCT AAG ATT AAA TTA ACA TTT ATT GGT CAT GGT AAA GAT 1968
Ser Asp Arg Pro Lys He Lys Leu Thr Phe He Gly His Gly Lys Asp 645 650 655 GAA TTT AAT ACT GAT ATA TTT GCA GGT TTT GAT GTA GAT TCA TTA TCC 2016
Glu Phe Asn Thr Asp He Phe Ala Gly Phe Asp Val Asp Ser Leu Ser 660 665 670
ACA GAA ATA GAA GCA GCA ATA GAT TTA GCT AAA GAG GAT ATT TCT CCT 2064 Thr Glu He Glu Ala Ala He Asp Leu Ala Lys Glu Asp He Ser Pro 675 680 685
AAG TCA ATA GAA ATA AAT TTA TTA GGA TGT AAT ATG TTT AGC TAC TCT 2112
Lys Ser He Glu He Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700
ATC AAC GTA GAG GAG ACT TAT CCT GGA AAA TTA TTA CTT AAA GTT AAA 2160
He Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys
705 710 715 ' 720
GAT AAA ATA TCA GAA TTA ATG CCA TCT ATA AGT CAA GAC TCT ATT ATA 2208
Asp Lys He Ser Glu Leu Met Pro Ser He Ser Gin Asp Ser He He 725 730 735 GTA AGT GCA AAT CAA TAT GAA GTT AGA ATA AAT AGT GAA GGA AGA AGA 2256
Val Ser Ala Asn Gin Tyr Glu Val Arg He Asn Ser Glu Gly Arg Arg 740 745 750
24: GAA TTA TTG GAT CAT TCT GGT GAA TGG ATA AAT AAA GAA GAA AGT ATT 2304 Glu Leu Leu Asp His Ser Gly Glu Trp He Asn Lys Glu Glu Ser He 755 760 765
ATA AAG GAT ATT TCA TCA AAA GAA TAT ATA TCA TTT AAT CCT AAA GAA 2352 He Lys Asp He Ser Ser Lys Glu Tyr He Ser Phe Asn Pro Lys Glu 770 775 780
AAT AAA ATT ACA GTA AAA TCT AAA AAT TTA CCT GAG CTA TCT ACA TTA 2400 Asn Lys He Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800
TTA CAA GAA ATT AGA AAT AAT TCT AAT TCA AGT GAT ATT GAA CTA GAA 2448 Leu Gin Glu He Arg Asn Asn Ser Asn Ser Ser Asp He Glu Leu Glu 805 810 815
GAA AAA GTA ATG TTA ACA GAA TGT GAG ATA AAT GTT ATT TCA AAT ATA 2496 Glu Lys Val Met Leu Thr Glu Cys Glu He Asn Val He Ser Asn He 820 825 830
GAT ACG CAA ATT GTT GAG GAA AGG ATT GAA GAA GCT AAG AAT TTA ACT 2544 Asp Thr Gin He Val Glu Glu Arg He Glu Glu Ala Lys Asn Leu Thr 835 840 845 TCT GAC TCT ATT AAT TAT ATA AAA GAT GAA TTT AAA CTA ATA GAA TCT 2592 Ser Asp Ser He Asn Tyr He Lys Asp Glu Phe Lys Leu He Glu Ser 850 855 860
ATT TCT GAT GCA CTA TGT GAC TTA AAA CAA CAG AAT GAA TTA GAA GAT 2640 He Ser Asp Ala Leu Cys Asp Leu Lys Gin Gin Asn Glu Leu Glu Asp 865 870 875 880
TCT CAT TTT ATA TCT TTT GAG GAC ATA TCA GAG ACT GAT GAG GGA TTT 2688 Ser His Phe He Ser Phe Glu Asp He Ser Glu Thr Asp Glu Gly Phe 885 890 895
AGT ATA AGA TTT ATT AAT AAA GAA ACT GGA GAA TCT ATA TTT GTA GAA 2736
Ser He Arg Phe He Asn Lys Glu Thr Gly Glu Ser He Phe Val Glu
900 905 910
ACT GAA AAA ACA ATA TTC TCT GAA TAT GCT AAT CAT ATA ACT GAA GAG 2784
Thr Glu Lys Thr He Phe Ser Glu Tyr Ala Asn His He Thr Glu Glu 915 920 925 ATT TCT AAG ATA AAA GGT ACT ATA TTT GAT ACT GTA AAT GGT AAG TTA 2832 He Ser Lys He Lys Gly Thr He Phe Asp Thr Val Asn Gly Lys Leu 930 ' ' 935 940
GTA AAA AAA GTA AAT TTA GAT ACT ACA CAC GAA GTA AAT ACT TTA AAT 2880 Val Lys Lvs Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 ' 950 955 960
GCT GCA TTT TTT ATA CAA TCA TTA ATA GAA TAT AAT AGT TCT AAA GAA 2928 Ala Ala Phe Phe He Gin Ser Leu He Glu Tyr Asn Ser Ser Lys Glu 965 970 975
TCT CTT AGT AAT TTA AGT GTA GCA ATG AAA GTC CAA GTT TAC GCT CAA 2976 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gin Val Tyr Ala Gin 980 985 990
TTA TTT AGT ACT GGT TTA AAT ACT ATT ACA GAT GCA GCC AAA GTT GTT 3024 Leu Phe Ser Thr Gly Leu Asn Thr He Thr Asp Ala Ala Lys Val Val 995 1000 1005 GAA TTA GTA TCA ACT GCA TTA GAT GAA ACT ATA GAC TTA CTT CCT ACA 3072
Glu Leu Val Ser Thr Ala Leu Asp Glu Thr He Asp Leu Leu Pro Thr 1010 1015 1020 TTA TCT GAA GGA TTA CCT ATA ATT GCA ACT ATT ATA GAT GGT GTA AGT 3120
Leu Ser Glu Gly Leu Pro He He Ala Thr He He Asp Gly Val Ser 1025 1030 1035 1040
TTA GGT GCA GCA ATC AAA GAG CTA AGT GAA ACG AGT GAC CCA TTA TTA 3168 Leu Gly Ala Ala He Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu
1045 1050 1055
AGA CAA GAA ATA GAA GCT AAG ATA GGT ATA ATG GCA GTA AAT TTA ACA 3216
Arg Gin Glu He Glu Ala Lys He Gly He Met Ala Val Asn Leu Thr 1060 1065 1070
ACA GCT ACA ACT GCA ATC ATT ACT TCA TCT TTG GGG ATA GCT AGT GGA 3264
Thr Ala Thr Thr Ala He He Thr Ser Ser Leu Gly He Ala Ser Gly 1075 1080 1085
TTT AGT ATA CTT TTA GTT CCT TTA GCA GGA ATT TCA GCA GGT ATA CCA 3312
Phe Ser He Leu Leu Val Pro Leu Ala Gly He Ser Ala Gly He Pro 1090 1095 1100 AGC TTA GTA AAC AAT GAA CTT GTA CTT CGA GAT AAG GCA ACA AAG GTT 3360
Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val 1105 1110 1115 1120
GTA GAT TAT TTT AAA CAT GTT TCA TTA GTT GAA ACT GAA GGA GTA TTT 3408 Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe
1125 1130 1135
ACT TTA TTA GAT GAT AAA ATA ATG ATG CCA CAA GAT GAT TTA GTG ATA 3456
Thr Leu Leu Asp Asp Lys He Met Met Pro Gin Asp Asp Leu Val He 1140 ' 1145 1150
TCA GAA ATA GAT TTT AAT AAT AAT TCA ATA GTT TTA GGT AAA TGT GAA 3504
Ser Glu He Asp Phe Asn Asn Asn Ser He Val Leu Gly Lys Cys Glu 1155 1160 1165
ATC TGG AGA ATG GAA GGT GGT TCA GGT CAT ACT GTA ACT GAT GAT ATA 3552
He Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp He 1170 1175 1180 GAT CAC TTC TTT TCA GCA CCA TCA ATA ACA TAT AGA GAG CCA CAC TTA 3600
Asp His Phe Phe Ser Ala Pro Ser He Thr Tyr Arg Glu Pro His Leu 1185 1190 1195 1200
TCT ATA TAT GAC GTA TTG GAA GTA CAA AAA GAA GAA CTT GAT TTG TCA 3648 Ser He Tyr Asp Val Leu Glu Val Gin Lys Glu Glu Leu Asp Leu Ser
1205 1210 1215
AAA GAT TTA ATG GTA TTA CCT AAT GCT CCA AAT AGA GTA TTT GCT TGG 3696
Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230
GAA ACA GGA TGG ACA CCA GGT TTA AGA AGC TTA GAA AAT GAT GGC ACA 3744
Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245
AAA CTG TTA GAC CGT ATA AGA GAT AAC TAT GAA GGT GAG TTT TAT TGG 3792
Lys Leu Leu Asp Arg He Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 AGA TAT TTT GCT TTT ATA GCT GAT GCT TTA ATA ACA ACA TTA AAA CCA 3840
Arg Tyr Phe Ala Phe He Ala Asp Ala Leu He Thr Thr Leu Lys Pro 1265 1270 1275 1280 AGA TAT GAA GAT ACT AAT ATA AGA ATA AAT TTA GAT AGT AAT ACT AGA 3888 Arg Tyr Glu Asp Thr Asn He Arg He Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295 AGT TTT ATA GTT CCA ATA ATA ACT ACA GAA TAT ATA AGA GAA AAA TTA 3936 Ser Phe He Val Pro He He Thr Thr Glu Tyr He Arg Glu Lys Leu 1300 1305 1310
TCA TAT TCT TTC TAT GGT TCA GGA GGA ACT TAT GCA TTG TCT CTT TCT 3984 Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 * 1325
CAA TAT AAT ATG GGT ATA AAT ATA GAA TTA AGT GAA AGT GAT GTT TGG 4032 Gin Tyr Asn Met Gly He Asn He Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340
ATT ATA GAT GTT GAT AAT GTT GTG AGA GAT GTA ACT ATA GAA TCT GAT 4080 He He Asp Val Asp Asn Val Val Arg Asp Val Thr He Glu Ser Aεp 1345 1350 1355 1360
AAA ATT AAA AAA GGT GAT TTA ATA GAA GGT ATT TTA TCT ACA CTA AGT 4128 Lys He Lys Lys Gly Asp Leu He Glu Gly He Leu Ser Thr Leu Ser 1365 1370 1375 ATT GAA GAG AAT AAA ATT ATC TTA AAT AGC CAT GAG ATT AAT TTT TCT 4176 He Glu Glu Asn Lys He He Leu Asn Ser His Glu He Asn Phe Ser 1380 1385 1390
GGT GAG GTA AAT GGA AGT AAT GGA TTT GTT TCT TTA ACA TTT TCA ATT 4224 Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser He 1395 1400 1405
TTA GAA GGA ATA AAT GCA ATT ATA GAA GTT GAT TTA TTA TCT AAA TCA 4272 Leu Glu Gly He Asn Ala He He Glu Val Asp Leu Leu Ser Lys Ser 1410 1415 1420
TAT AAA TTA CTT ATT TCT GGC GAA TTA AAA ATA TTG ATG TTA AAT TCA 4320 Tyr Lys Leu Leu He Ser Gly Glu Leu Lys He Leu Met Leu Asn Ser 1425 1430 1435 1440
AAT CAT ATT CAA CAG AAA ATA GAT TAT ATA GGA TTC AAT AGC GAA TTA 4368
Asn His He Gin Gin Lys He Asp Tyr He Gly Phe Asn Ser Glu Leu
1445 1450 1455 CAG AAA AAT ATA CCA TAT AGC TTT GTA GAT AGT GAA GGA AAA GAG AAT 4416 Gin Lys Asn He Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 1470
GGT TTT ATT AAT GGT TCA ACA AAA GAA GGT TTA TTT GTA TCT GAA TTA 4464 Gly Phe He Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485
CCT GAT GTA GTT CTT ATA AGT AAG GTT TAT ATG GAT GAT AGT AAG CCT 4512 Pro Asp Val Val Leu He Ser Lys Val Tyr Met Asp Asp Ser Lys Pro 1490 1495 1500
TCA TTT GGA TAT TAT AGT AAT AAT TTG AAA GAT GTC AAA GTT ATA ACT 4560 Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Aεp Val Lyε Val He Thr 1505 1510 1515 1520
AAA GAT AAT GTT AAT ATA TTA ACA GGT TAT TAT CTT AAG GAT GAT ATA 4608 Lys Asp Asn Val Asn He Leu Thr Gly Tyr Tyr Leu Lys Asp Aεp He 1525 ' 1530 1535 AAA ATC TCT CTT TCT TTG ACT CTA CAA GAT GAA AAA ACT ATA AAG TTA 4656 Lyε He Ser Leu Ser Leu Thr Leu Gin Asp Glu Lys Thr He Lys Leu 1540 1545 1550 AAT AGT GTG CAT TTA GAT GAA AGT GGA GTA GCT GAG ATT TTG AAG TTC 4704
Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu He Leu Lys Phe 1555 1560 1565
ATG AAT AGA AAA GGT AAT ACA AAT ACT TCA GAT TCT TTA ATG AGC TTT 4752
Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580
TTA GAA AGT ATG AAT ATA AAA AGT ATT TTC GTT AAT TTC TTA CAA TCT 4800
Leu Glu Ser Met Asn He Lys Ser He Phe Val Asn Phe Leu Gin Ser 1585 1590 1595 1600
AAT ATT AAG TTT ATA TTA GAT GCT AAT TTT ATA ATA AGT GGT ACT ACT 4848
Asn He Lys Phe He Leu Asp Ala Asn Phe He He Ser Gly Thr Thr
1605 1610 1615
TCT ATT GGC CAA TTT GAG TTT ATT TGT GAT GAA AAT GAT AAT ATA CAA 4896
Ser He Gly Gin Phe Glu Phe He Cys Asp Glu Asn Asp Asn He Gin
1620 1625 1630
CCA TAT TTC ATT AAG TTT AAT ACA CTA GAA ACT AAT TAT ACT TTA TAT 4944
Pro Tyr Phe He Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645 GTA GGA AAT AGA CAA AAT ATG ATA GTG GAA CCA AAT TAT GAT TTA GAT 4992
Val Gly Asn Arg Gin Asn Met He Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660
GAT TCT GGA GAT ATA TCT TCA ACT GTT ATC AAT TTC TCT CAA AAG TAT 5040 Asp Ser Gly Asp He Ser Ser Thr Val He Asn Phe Ser Gin Lys Tyr 1665 1670 1675 1680
CTT TAT GGA ATA GAC AGT TGT GTT AAT AAA GTT GTA ATT TCA CCA AAT 5088
Leu Tyr Gly He Asp Ser Cys Val Asn Lys Val Val He Ser Pro Asn * 1685 1690 1695
ATT TAT ACA GAT GAA ATA AAT ATA ACG CCT GTA TAT GAA ACA AAT AAT 5136
He Tyr Thr Asp Glu He Asn He Thr Pro Val Tyr Glu Thr Asn Asn
1700 1705 1710
ACT TAT CCA GAA GTT ATT GTA TTA GAT GCA AAT TAT ATA AAT GAA AAA 5184
Thr Tyr Pro Glu Val He Val Leu Asp Ala Asn Tyr He Asn Glu Lys 1715 1720 1725 ATA AAT GTT AAT ATC AAT GAT CTA TCT ATA CGA TAT GTA TGG AGT AAT 5232
He Asn Val Asn He Asn Asp Leu Ser He Arg Tyr Val Trp Ser Asn 1730 1735 " 1740
GAT GGT AAT GAT TTT ATT CTT ATG TCA ACT AGT GAA GAA AAT AAG GTG 5280 Asp Gly Asn Asp Phe He Leu Met Ser Thr Ser Glu Glu Asn Lys Val 1745 1750 1755 1760
TCA CAA GTT AAA ATA AGA TTC GTT AAT GTT TTT AAA GAT AAG ACT TTG 5328
Ser Gin Val Lys He Arg Phe Val Asn Val Phe Lys Aεp Lys Thr Leu 1765 1770 1775
GCA AAT AAG CTA TCT TTT AAC TTT AGT GAT AAA CAA GAT GTA CCT GTA 5376
Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gin Asp Val Pro Val
1780 1785 1790
AGT GAA ATA ATC TTA TCA TTT ACA CCT TCA TAT TAT GAG GAT GGA TTG 5424 Ser Glu He He Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805 ATT GGC TAT GAT TTG GGT CTA GTT TCT TTA TAT AAT GAG AAA TTT TAT 5472 He Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820 ATT AAT AAC TTT GGA ATG ATG GTA TCT GGA TTA ATA TAT ATT AAT GAT 5520 He Asn Asn Phe Gly Met Met Val Ser Gly Leu He Tyr He Asn Asp 1825 1830 1835 1840 TCA TTA TAT TAT TTT AAA CCA CCA GTA AAT AAT TTG ATA ACT GGA TTT 5568 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu He Thr Gly Phe 1845 ' 1850 1855
GTG ACT GTA GGC GAT GAT AAA TAC TAC TTT AAT CCA ATT AAT GGT GGA 5616 Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro He Asn Gly Gly
1860 1865 1870
GCT GCT TCA ATT GGA GAG ACA ATA ATT GAT GAC AAA AAT TAT TAT TTC 5664 Ala Ala Ser He Gly Glu Thr He He Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885
AAC CAA AGT GGA GTG TTA CAA ACA GGT GTA TTT AGT ACA GAA GAT GGA 5712
Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900
TTT AAA TAT TTT GCC CCA GCT AAT ACA CTT GAT GAA AAC CTA GAA GGA 5760
Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly
1905 * 1910 1915 1920 GAA GCA ATT GAT TTT ACT GGA AAA TTA ATT ATT GAC GAA AAT ATT TAT 5808 Glu Ala He Asp Phe Thr Gly Lys Leu He He Asp Glu Asn He Tyr 1925 1930 1935
TAT TTT GAT GAT AAT TAT AGA GGA GCT GTA GAA TGG AAA GAA TTA GAT 5856 Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp
1940 1945 ' 1950
GGT GAA ATG CAC TAT TTT AGC CCA GAA ACA GGT AAA GCT TTT AAA GGT 5904 Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly ' 1955 1960 1965
CTA AAT CAA ATA GGT GAT TAT AAA TAC TAT TTC AAT TCT GAT GGA GTT 5952 Leu Asn Gin He Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980
ATG CAA AAA GGA TTT GTT AGT ATA AAT GAT AAT AAA CAC TAT TTT GAT 6000 Met Gin Lvs Gly Phe Val Ser He Asn Asp Asn Lys Hiε Tyr Phe Asp 1985 ' 1990 1995 2000 GAT TCT GGT GTT ATG AAA GTA GGT TAC ACT GAA ATA GAT GGC AAG CAT 6048 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu He Asp Gly Lys His 2005 2010 2015
TTC TAC TTT GCT GAA AAC GGA GAA ATG CAA ATA GGA GTA TTT AAT ACA 6096 Phe Tyr Phe Ala Glu Asn Gly Glu Met Gin He Gly Val Phe Aεn Thr
2020 2025 2030
GAA GAT GGA TTT AAA TAT TTT GCT CAT CAT AAT GAA GAT TTA GGA AAT 6144 Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 2040 2045
GAA GAA GGT GAA GAA ATC TCA TAT TCT GGT ATA TTA AAT TTC AAT AAT 6192 Glu Glu Gly Glu Glu He Ser Tyr Ser Gly He Leu Asn Phe Asn Asn 2050 ' 2055 2060
AAA ATT TAC TAT TTT GAT GAT TCA TTT ACA GCT GTA GTT GGA TGG AAA 6240 Lys He Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080 GAT TTA GAG GAT GGT TCA AAG TA.T TAT TTT GAT GAA GAT ACA GCA GAA 6288 Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu 2085 2090 2095 GCA TAT ATA GGT TTG TCA TTA ATA AAT GAT GGT CAA TAT TAT TTT AAT 6336
Ala Tyr He Gly Leu Ser Leu He Asn Asp Gly Gin Tyr Tyr Phe Asn
2100 2105 2110 GAT GAT GGA ATT ATG CAA GTT GGA TTT GTC ACT ATA AAT GAT AAA GTC 6384
Asp Asp Gly He Met Gin Val Gly Phe Val Thr He Asn Asp Lys Val
2115 2120 2125
TTC TAC TTC TCT GAC TCT GGA ATT ATA GAA TCT GGA GTA CAA AAC ATA 6432 Phe Tyr Phe Ser Asp Ser Gly He He Glu Ser Gly Val Gin Asn He
2130 2135 2140
GAT GAC AAT TAT TTC TAT ATA GAT GAT AAT GGT ATA GTT CAA ATT GGT 6480
Asp Asp Asn Tyr Phe Tyr He Asp Asp Asn Gly He Val Gin He Gly 2145 2150 2155 2160
GTA TTT GAT ACT TCA GAT GGA TAT AAA TAT TTT GCA CCT GCT AAT ACT 6528
Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr
2165 2170 2175
GTA AAT GAT AAT ATT TAC GGA CAA GCA GTT GAA TAT AGT GGT TTA GTT 6576
Val Asn Asp Asn He Tyr Gly Gin Ala Val Glu Tyr Ser Gly Leu Val
2180 2185 2190 AGA GTT GGG GAA GAT GTA TAT TAT TTT GGA GAA ACA TAT ACA ATT GAG 6624
Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr He Glu
2195 2200 2205
ACT GGA TGG ATA TAT GAT ATG GAA AAT GAA AGT GAT AAA TAT TAT TTC 6672 Thr Gly Trp He Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe
2210 2215 2220
AAT CCA GAA ACT AAA AAA GCA TGC AAA GGT ATT AAT TTA ATT GAT GAT 6720
Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly He Asn Leu He Asp Asp 2225 2230 ' 2235 2240
ATA AAA TAT TAT TTT GAT GAG AAG GGC ATA ATG AGA ACG GGT CTT ATA 6768
He Lys Tyr Tyr Phe Asp Glu Lys Gly He Met Arg Thr Gly Leu He
2245 2250 2255
TCA TTT GAA AAT AAT AAT TAT TAC TTT AAT GAG AAT GGT GAA ATG CAA 6816
Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin
2260 2265 2270 TTT GGT TAT ATA AAT ATA GAA GAT AAG ATG TTC TAT TTT GGT GAA GAT 6864
Phe Gly Tyr He Asn He Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp
2275 2280 ' 2285
GGT GTC ATG CAG ATT GGA GTA TTT AAT ACA CCA GAT GGA TTT AAA TAC 6912 Gly Val Met Gin He Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr
2290 2295 2300
TTT GCA CAT CAA AAT ACT TTG GAT GAG AAT TTT GAG GGA GAA TCA ATA 6960
Phe Ala His Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser He 2305 2310 2315 2320
AAC TAT ACT GGT TGG TTA GAT TTA GAT GAA AAG AGA TAT TAT TTT ACA 7008
Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr
2325 2330 ' 2335
GAT GAA TAT ATT GCA GCA ACT GGT TCA GTT ATT ATT GAT GGT GAG GAG 7056
Asp Glu Tyr He Ala Ala Thr Gly Ser Val He He Asp Gly Glu Glu
2340 2345 2350 TAT TAT TTT GAT CCT GAT ACA GCT CAA TTA GTG ATT AGT GAA 7098
Tyr Tyr Phe Asp Pro Asp Thr Ala Gin Leu Val He Ser Glu
2355 2360 2365
TAG 7101 (2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2366 amino acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Met Ser Leu Val Asn Arg Lys Gin Leu Glu Lys Met Ala Asn Val Arg 1 5 10 15 Phe Arg Thr Gin Glu Asp Glu Tyr Val Ala He Leu Asp Ala Leu Glu
20 25 30
Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys
35 40 45
Leu Lyε Asp He Asn Ser Leu Thr Asp He Tyr He Aεp Thr Tyr Lys
50 55 60
Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val
65 70 * 75 80
Thr Glu Val Leu Glu Leu Lys Asn Aεn Asn Leu Thr Pro Val Glu Lyε 85 90 95
Asn Leu Hr: Phe Val Trp He Gly Gly Gin He Asn Asp Thr Ala He
100 105 110
Asn Tyr He Asn Gin Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn
115 120 125
Val Phe Tyr Asp Ser Aεn Ala Phe Leu He Asn Thr Leu Lys Lys Thr
130 135 140
Val Val Glu Ser Ala He Asn Aεp Thr Leu Glu Ser Phe Arg Glu Asn 145 150 155 160
Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met
165 170 175 Glu He He Tyr Asp Lys Gin Lys Asn Phe He Asn Tyr Tyr Lys Ala
180 185 190
Gin Arg Glu Glu Asn Pro Glu Leu He He Asp Asp He Val Lys Thr
195 200 205
Tyr Leu Ser Asn Glu Tyr Ser Lys Glu He Asp Glu Leu Asn Thr Tyr
210 215 220
He Glu Glu Ser Leu Asn Lys He Thr Gin Asn Ser Gly Asn Asp Val 225 230 235 240
Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu
245 250 255 Gin Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp He Leu
260 265 270
Arg He Ser Ala Leu Lys Glu He Gly Gly Met Tyr Leu Asp Val Asp 275 280 285
Met Leu Pro Gly He Gin Pro Asp Leu Phe Glu Ser He Glu Lys Pro 290 295 300
Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala He 305 310 315 320 Met Lys Tyr Lys Glu Tyr He Pro Glu Tyr Thr Ser Glu His Phe Asp 325 330 335
Met Leu Asp Glu Glu Val Gin Ser Ser Phe Glu Ser Val Leu Ala Ser 5 340 345 350
Lys Ser Asp Lys Ser Glu He Phe Ser Ser Leu Gly Asp Met Glu Ala 355 360 365
10 Ser Pro Leu Glu Val Lys He Ala Phe Asn Ser Lys Gly He He Asn 370 375 380
Gin Gly Leu He Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu He Val 385 390 395 400
15
Lys Gin He Glu Asn Arg Tyr Lys He Leu Asn Asn Ser Leu Asn Pro 405 410 415
Ala He Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe He 20 420 425 430
Asp Ser He Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met 435 440 445
25 Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr 450 455 460
Thr He Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gin Asp 465 470 475 480
30
Leu Leu Met Phe Lys Glu Gly Ser Met Asn He His Leu He Glu Ala 485 490 495
Asp Leu Arg Asn Phe Glu He Ser Lys Thr Asn He Ser Gin Ser Thr
J.i 500 505 510
Glu Gin Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Ly∑ 515 520 525
40 Ala Gin Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly 530 535 540
Glu Asp Asp Asn Leu Asp Phe Ser Gin Asn He Val Val Asp Lys Glu 545 550 555 560
45
Tyr Leu Leu Glu Lys He Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly 565 570 575
Tyr He His Tyr He Val Gin Leu Gin Gly Asp Lys He Ser Tyr Glu 50 580 585 590
Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe 595 600 605
55 Gin Lys Asn He Glu Asp Ser Glu He Ala Tyr Tyr Tyr Asn Pro Gly 610 615 620
Asp Gly Glu He Gin Glu He Asp Lys Tyr Lys He Pro Ser He He
625 630 635 640
60
Ser Asp A g Pro Lys He Lys Leu Thr Phe He Gly His Gly Lys Asp
645 650 ' 655 "
Glu Phe Asn Thr Asp He Phe Ala Gly Phe Asp Val Asp Ser Leu Ser
65 660 665 670
Thr Glu He Glu Ala Ala He Asp Leu Ala Lys Glu Asp He Ser Pro 675 680 685 Lys Ser He Glu He Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser 690 695 700
He Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lvs 705 710 715 720
Asp Lys He Ser Glu Leu Met Pro Ser He Ser Gin Asp Ser He He 725 730 735 Val Ser Ala Asn Gin Tyr Glu Val Arg He Asn Ser Glu Gly Arg Arg
740 745 750
Glu Leu Leu Asp His Ser Gly Glu Trp He Asn Lys Glu Glu Ser He 755 760 765
He Lys Asp He Ser Ser Lys Glu Tyr He Ser Phe Asn Pro Lys Glu 770 775 780
Asn Lys He Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu 785 790 795 800
Leu Gin Glu He Arg Asn Asn Ser Asn Ser Ser Asp He Glu Leu Glu 805 810 815 Glu Lys Val Met Leu Thr Glu Cys Glu He Asn Val He Ser Asn He
820 825 830
Asp Thr Gin He Val Glu Glu Arg He Glu Glu Ala Lys Asn Leu Thr 835 840 845
Ser Asp Ser He Asn Tyr He Lys Asp Glu Phe Lys Leu He Glu Ser 850 855 860
He Ser Asp Ala Leu Cys Asp Leu Lys Gin Gin Asn Glu Leu Glu Asp 865 870 875 880
Ser His Phe He Ser Phe Glu Asp He Ser Glu Thr Asp Glu Gly Phe 885 890 895 Ser He Arg Phe He Asn Lyε Glu Thr Gly Glu Ser He Phe Val Glu
900 905 910
Thr Glu Lys Thr He Phe Ser Glu Tyr Ala Asn His He Thr Glu Glu 915 920 925
He Ser Lys He Lys Gly Thr He Phe Asp Thr Val Asn Gly Lys Leu 930 935 940
Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn 945 ' ' 950 955 960
Ala Ala Phe Phe He Gin Ser Leu He Glu Tyr Asn Ser Ser Lys Glu 965 970 975 Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gin Val Tyr Ala Gin
980 985 990
Leu Phe Ser Thr Gly Leu Asn Thr He Thr Asp Ala Ala Lys Val Val 995 1000 1005
Glu Leu Val Ser Thr Ala Leu Asp Glu Thr He Asp Leu Leu Pro Thr 1010 1015 1020
Leu Ser Glu Gly Leu Pro He He Ala Thr He He Asp Gly Val Ser 1025 1030 1035 1040
Leu Gly Ala Ala He Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu 1045 ' 1050 1055 Arg Gin Glu He Glu Ala Lys He Gly He Met Ala Val Asn Leu Thr 1060 1065 1070
Thr Ala Thr Thr Ala He He Thr Ser Ser Leu Gly He Ala Ser Gly 1075 1080 1085
Phe Ser He Leu Leu Val Pro Leu Ala Gly He Ser Ala Gly He Pro 1090 1095 1100
Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val
1105 1110 1115 1120
Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe 1125 1130 1135
Thr Leu Leu Asp Asp Lys He Met Met Pro Gin Asp Asp Leu Val He 1140 1145 1150
Ser Glu He Asp Phe Asn Asn Asn Ser He Val Leu Gly Lys Cys Glu 1155 1160 1165
He Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp He 1170 1175 1180
Asp His Phe Phe Ser Ala Pro Ser He Thr Tyr Arg Glu Pro His Leu
1185 1190 1195 1200
Ser He Tyr Asp Val Leu Glu Val Gin Lys Glu Glu Leu Asp Leu Ser 1205 1210 1215
Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp 1220 1225 1230
Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr 1235 1240 1245
Lys Leu Leu Asp Arg He Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp 1250 1255 1260 Arg Tyr Phe Ala Phe He Ala Asp Ala Leu He Thr Thr Leu Lys Pro 1265 1270 * 1275 1280
Arg Tyr Glu Asp Thr Asn He Arg He Asn Leu Asp Ser Asn Thr Arg 1285 1290 1295
Ser Phe He Val Pro He He Thr Thr Glu Tyr He Arg Glu Lyε Leu 1300 1305 1310
Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser 1315 1320 1325
Gin Tyr Asn Met Gly He Asn He Glu Leu Ser Glu Ser Asp Val Trp 1330 1335 1340 He He Asp Val Asp Asn Val Val Arg Asp Val Thr He Glu Ser Asp
1345 1350 1355 1360
Lys He Lys Lys Gly Asp Leu He Glu Gly He Leu Ser Thr Leu Ser 1365 1370 1375
He Glu Glu Asn Lys He He Leu Asn Ser His Glu He Asn Phe Ser 1380 1385 1390
Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser He 1395 1400 1405
Leu Glu Gly He Asn Ala He He Glu Val Asp Leu Leu Ser Lys Ser
1410 1415 1420 Tyr Lys Leu Leu He Ser Gly Glu Leu Lys He Leu Met Leu Asn Ser 1425 1430 1435 1440
Asn His He Gin Gin Lys He Asp Tyr He Gly Phe Asn Ser Glu Leu 1445 1450 1455
Gin Lys Asn He Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn 1460 1465 ' 1470
Gly Phe He Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu 1475 1480 1485
Pro Asp Val Val Leu He Ser Lys Val Tyr Met Asp Asp Ser Lvs Pro 1490 1495 1500
Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val He Thr 1505 1510 1515 1520
Lys Asp Asn Val Asn He Leu Thr Gly Tyr Tyr Leu Lys Asp Asp He 1525 1530 1535
Lyε He Ser Leu Ser Leu Thr Leu Gin Asp Glu Lyε Thr He Lys Leu 1540 1545 1550
Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu He Leu Lyε Phe 1555 1560 1565
Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe 1570 1575 1580
Leu Glu Ser Met Asn He Lys Ser He Phe Val Asn Phe Leu Gin Ser 1585 1590 1595 1600
Asn He Lys Phe He Leu Asp Ala Asn Phe He He Ser Gly Thr Thr 1605 1610 1615
Ser He Gly Gin Phe Glu Phe He Cys Asp Glu Asn Asp Asn He Gin 1620 1625 1630 Pro Tyr Phe He Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr 1635 1640 1645
Val Gly Asn Arg Gin Asn Met He Val Glu Pro Asn Tyr Asp Leu Asp 1650 1655 1660
Asp Ser Gly Asp He Ser Ser Thr Val He Asn Phe Ser Gin Lys Tyr 1665 1670 1675 ' 1680
Leu Tyr Gly He Asp Ser Cys Val Aεn Lys Val Val He Ser Pro Asn 1685 1690 1695
He Tyr Thr Asp Glu He Asn He Thr Pro Val Tyr Glu Thr Asn Asn 1700 1705 1710 Thr Tyr Pro Glu Val He Val Leu Asp Ala Asn Tyr He Asn Glu Lys 1715 1720 1725
He Asn Val Asn He Asn Asp Leu Ser He Arg Tyr Val Trp Ser Asn 1730 1735 1740
Asp Gly Asn Asp Phe He Leu Met Ser Thr Ser Glu Glu Asn Lys Val
1745 1750 1755 ' 1760
Ser Gin Val Lys He Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu 1765 1770 1775
Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gin Asp Val Pro Val
1780 1785 1790 Ser Glu He He Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu 1795 1800 1805
He Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr 1810 1815 1820
He Asn Asn Phe Gly Met Met Val Ser Gly Leu He Tyr He Asn Asp 1825 1830 1835 1840 Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu He Thr Gly Phe
1845 1850 1855
Val Thr Val Gly Aεp Asp Lys Tyr Tyr Phe Asn Pro He Asn Gly Gly 1860 1865 1870
Ala Ala Ser He Gly Glu Thr He He Asp Asp Lys Asn Tyr Tyr Phe 1875 1880 1885
Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe Ser Thr Glu Asp Gly 1890 1895 1900
Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly 1905 1910 1915 1920
Glu Ala He Asp Phe Thr Gly Lys Leu He He Asp Glu Asn He Tyr 1925 1930 1935
Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp 1940 1945 1950
Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly 1955 1960 1965
Leu Asn Gin He Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val 1970 1975 1980
Met Gin Lys Gly Phe Val Ser He Asn Asp Asn Lys His Tyr Phe Asp 1985 ' ' 1990 1995 2000 Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu He Asp Gly Lys His
2005 2010 2015
Phe Tyr Phe Ala Glu Asn Gly Glu Met Gin He Gly Val Phe Asn Thr 2020 2025 2030
Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn 2035 ' ' 2040 2045
Glu Glu Gly Glu Glu He Ser Tyr Ser Gly He Leu Asn Phe Asn Asn 2050 2055 2060
Lys He Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys 2065 2070 2075 2080 Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu
2085 2090 2095
Ala Tyr He Gly Leu Ser Leu He Asn Asp Gly Gin Tyr Tyr Phe Asn 2100 2105 ' 2110
Asp Asp Gly He Met Gin Val Gly Phe Val Thr He Asn Asp Lys Val 2115 2120 2125
Phe Tyr Phe Ser Asp Ser Gly He He Glu Ser Gly Val Gin Asn He 2130 2135 2140
Asp Asp Asn Tyr Phe Tyr He Asp Asp Asn Gly He Val Gin He Gly 2145 ' 2150 2155 2160 Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr 2165 2170 !175
Val Asn Aεp Asn He Tyr Gly Gin Ala Val Glu Tyr Ser Gly Leu Val 2180 2185 2190
Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr lie Glu 2195 2200 2205
Thr Gly Trp He Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe 2210 2215 2220
Asn Pro Glu Thr Lys Lys Ala Cys Lyε Gly He Asn -Leu He Asp Asp 2230 ' 2235 2240 He Lys Tyr Tyr Phe Asp Glu Lys Gly He Met Arg Thr Gly Leu He
2245 2250 2255
Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gin 2260 2265 2270
Phe Gly Tyr He Asn He Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp 2275 2280 2285
Gly Val Met Gin He Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr 2290 2295 2300
Phe Ala His Gin Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser He 2305 2310 2315 2320
Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr 2325 2330 2335
Asp Glu Tyr He Ala Ala Thr Gly Ser Val He He Asp Gly Glu Glu 2340 2345 2350
Tyr Tyr Phe Asp Pro Asp Thr Ala Gin Leu Val He Ser Glu 2355 2360 2365
(2) INFORMATION FOR SEQ ID NO : 11 :
( l ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(u) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION. SEQ ID NO : 11
TAGAAAAAAT GGCAAATGT
(2) INFORMATION FOR SEQ ID NO: 12:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12. TTTCATCTTG TAGAGTCAAA G
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13 GATGCCACAA GATGATTTAG TG (2) INFORMATION FOR SEQ ID NO: 14:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(n) MOLECULE TYPE; DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CTAATTGAGC TGTATCAGGA TC (2) INFORMATION FOR SEQ ID NO: 15: ill SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 15 CGGAATTCCT AGAAAAAATG GCAAATG (2) INFORMATION FOR SEQ ID NO: 16:
• i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ri) MOLECULE TYPE: DNA (genomic) (XI) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GCTCTAGAAT GACCATAAGC TAGCCA 26 (2) INFORMATION FOR SEQ ID NO: 17:
'l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ri) MOLECULE TYPE: DNA (genomic)
(κi) SEQUENCE DESCRIPTION: SEQ ID NO: 17
CGGAATTCGA GTTGGTAGAA AGGTGGA
(2) INFORMATION FOR SEQ ID NO: 18:
(r) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(u) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CGGAATTCGG TTATTATCTT AAGGATG 27
(2) INFORMATION FOR SEQ ID NO: 19:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear tii) MOLECULE TYPE: DNA (genomic) 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CGGAATTCTT GATAACTGGA TTTGTGAC 28 5 (2) INFORMATION FOR SEQ ID NO : 20 •
', l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 511 ammo acids
(B) TYPE: ammo acid 0 (C) STRANDEDNESS. unknown
(D) TOPOLOGY: unknown
(ll) MOLECULE TYPE: protem 5 (κi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
Leu He Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn 1 10 15 0 Pro He Asn Gly Gly Ala Ala Ser He Gly Glu Thr He He Asp Asp
20 25 30
Lys Asn Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val Phe 35 ' 40 45
Ser Thr Glu Asp Gly Phe Lyε Tyr Phe Ala Pro Ala Asn Thr Leu Asp 50 55 60
Glu Asn Leu Glu Gly Glu Ala He Asp Phe Thr Gly Lys Leu He He
50 65 70 75 80
Asp Glu Asn He Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu 85 90 95 n-ι Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly 100 105 110
Lys Ala Phe Lys Gly Leu Asn Gin He Gly Asp Tyr Lys Tyr Tyr Phe 115 120 125
60
Asn Ser Asp Gly Val Met Gin Lys Gly Phe Val Ser He Asn Asp Asn 130 135 L40
Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu 65 145 150 155 160
He Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gin He 165 170 175
70 Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn 180 185 190
Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu He Ser Tyr Ser Gly He 195 200 205
Leu Asn Phe Asn Asn Lys He Tyr Tyr Phe Asp Asp Ser Phe Thr Ala 210 215 ' 220
Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp 225 230 235 240
Glu Asp Thr Ala Glu Ala Tyr He Gly Leu Ser Leu He Asn Asp Gly 245 ' 25' 255
Gin Tyr Tyr Phe Asn Asp Asp Gly He Met Gin Val Gly Phe Val Thr 260 265 270
He Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly He He Glu Ser 275 280 285
Gly Val Gin Asn He Asp Asp Asn Tyr Phe Tyr He Asp Asp Asn Gly 290 295 300
He Val Gin He Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe 305 310 315 ' 320
Ala Pro Ala Asn Thr Val Asn Asp Aεn He Tyr Gly Gin Ala Val Glu 325 330 335 Tyr Ser Gly Leu Val Arg Val Gly Glu Aεp Val Tyr Tyr Phe Gly Glu
340 345 350
Thr Tyr Thr He Glu Thr Gly Trp He Tyr Asp Met Glu Asn Glu Ser 355 360 ' 365
Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly He 370 ' 375 380
Asn Leu He Asp Asp He Lys Tyr Tyr Phe Asp Glu Lys Gly He Met 385 390 ' 395 400
Arg Thr Gly Leu He Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu 405 410 415 Aεn Gly Glu Met Gin Phe Gly Tyr He Asn He Glu Asp Lys Met Phe
420 425 430
Tyr Phe Gly Glu Asp Gly Val Met Gin He Gly Val Phe Asn Thr Pro 435 440 445
Asp Gly Phe Lys Tyr Phe Ala His Gin Asn Thr Leu Asp Glu Aεn Phe 450 455 460
Glu Gly Glu Ser He Aεn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lyε 465 470 475 480
Arg Tyr Tyr Phe Thr Aεp Glu Tyr He Ala Ala Thr Gly Ser Val He 485 490 495
He Asp Gly Glu Glu Tyr Tyr Phe Aεp Pro Asp Thr Ala Gin Leu 500 * 505 510 INFORMATION FOR SEQ ID NO : 21 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 608 amino acids ( B ) TYPE : ammo ac id
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(n) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21.
Ser Glu Glu Asn Lys Val Ser Gin Val Lys He Arg Phe Val Asn Val 1 5 10 15
Phe Lys Asp Lys Thr Leu Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp 20 25 30
Lys Gin Asp Val Pro Val Ser Glu He He Leu Ser Phe Thr Pro Ser 35 40 45
Tyr Tyr Glu Asp Gly Leu He Gly Tyr Asp Leu Gly Leu Val Ser Leu 50 55 60 Tyr Aεn Glu Lys Phe Tyr He Asn Asn Phe Gly Met Met Val Ser Gly
65 70 75 80
Leu He Tyr He Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro Val Aεn 85 90 95
Asn Leu He Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe 100 105 110
Asn Pro He Asn Gly Gly Ala Ala Ser He Gly Glu Thr He He Asp 115 120 125
Asp Lys Asn Tyr Tyr Phe Asn Gin Ser Gly Val Leu Gin Thr Gly Val 130 * 135 140 Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu
145 150 155 160
Asp Glu Asn Leu Glu Gly Glu Ala He Asp Phe Thr Gly Lys Leu He 165 170 175
He Asp Glu Asn He Tyr Tyr Phe Asp Asn Tyr Arg Gly Ala Val 180 190
Glu Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr 195 ' 200 205
Gly Lys Ala Phe Lys Gly Leu Asn Gin He Gly Asp Tyr Lys Tyr Tyr
210 215 220 Phe Asn Ser Asp Gly Val Met Gin Lys Gly Phe Val Ser He Asn Asp
225 230 235 240
Asn Lyε His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr
245 250 255
Glu He Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gin
260 265 270
He Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His Hrs ' 275 280 285
Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu He Ser Tyr Ser Gly 290 295 300 He Leu Asn Phe Asn Asn Lys He Tyr Tyr Phe Asp Asp Ser Phe Thr
Figure imgf000263_0001
Glu Asn Gly Glu Met Gin Phe Glv Tyr He Asn He Glu Asp Lys Met 515 520 525
Phe Tyr Phe Gly Glu Asp Gly Val Met Gin He Gly Val Phe Asn Thr 530 535 540 Pro Asp Gly Phe Lys Tyr Phe Ala Hrs Gin Asn Thi Leu Asp Glu Asn
545 550 555 560
Phe Glu Gly Glu Ser He Asn Tyr Thr Glv Trp Leu Asp Leu Asp Glu 565 570 575
Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr He Ala Ala Thr Gly Sei Val 580 585 590
He He Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gin Leu 595 600 605 NFORMATION FOR SEQ ID NO 22
H) SEQUENCE CHARACTERISTICS (A) LENGTH 1330 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY linear (ii) MOLECULE TYPE DNA (genomic)
!ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 1314 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
ATG GCT CGT CTG CTG TCT ACC TTC ACT GAA TAC ATC AAG AAC ATC ATC 48 Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr He Lys Asn He He 1 5 10 15
AAT ACC TCC ATC CTG AAC CTG CGC TAC GAA TCC AAT CAC CTG ATC GAC 96
Asn Thr Ser He Leu Asn Leu Arg Tyr Glu Ser Asn His Leu He Asp 20 25 30
CTG TCT CGC TAC GCT TCC AAA ATC AAC ATC GGT TCT AAA GTT AAC TTC 144 Leu Ser Arg Tyr Ala Ser Lys He Asn He Gly Ser Lys Val Asn Phe 35 ' 40 45 GAT CCG ATC GAC AAG AAT CAG ATC CAG CTG TTC AAT CTG GAA TCT TCC 192 Asp Pro He Asp Lys Asn Gin He Gin Leu Phe Asn Leu Glu Ser Ser 50 55 60
AAA ATC GAA GTT ATC CTG AAG AAT GCT ATC GTA TAC AAC TCT ATG TAC 240 Lys He Glu Val He Leu Lys Asn Ala He Val Tyr Asn Ser Met Tyr 65 70 75 80
GAA AAC TTC TCC ACC TCC TTC TGG ATC CGT ATC CCG AAA TAC TTC AAC 288 Glu Asn Phe Ser Thr Ser Phe Trp He Arg He Pro Lys Tyr Phe Asn 85 90 95
TCC ATC TCT CTG AAC AAT GAA TAC ACC ATC ATC AAC TGC ATG GAA AAC 336 Ser He Ser Leu Aεn Asn Glu Tyr Thr He He Aεn Cys Met Glu Asn 100 105 ' 110
AAT TCT GGT TGG AAA GTA TCT CTG AAC TAC GGT GAA ATC ATC TGG ACT 384 Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu He He Trp Thr 115 " 120 125 CTG CAG GAC ACT CAG GAA ATC AAA CAG CGT GTT GTA TTC AAA TAC TCT 432 Leu Gin Asp Thr Gin Glu He Lys Gin Arg Val Val Phe Lys Tyr Ser 130 135 140
CAG ATG ATC AAC ATC TCT GAC TAC ATC AAT CGC TGG ATC TTC GTT ACC 480 Gin Met He Asn He Ser Asp Tyr He Asn Arg Trp He Phe Val Thr 145 150 155 160
ATC ACC AAC AAT CGT CTG AAT AAC TCC AAA ATC TAC ATC AAC GGC CGT 528 He Thr Asn Asn Arg Leu Asn Asn Ser Lys He Tyr He Asn Gly Arg 165 170 175
CTG ATC GAC CAG AAA CCG ATC TCC AAT CTG GGT AAC ATC CAC GCT TCT 576
Leu He Asp Gin Lys Pro He Ser Asn Leu Gly Asn He His Ala Ser 180 185 190
AAT AAC ATC ATG TTC AAA CTG GAC GGT TGT CGT GAC ACT CAC CGC TAC 624
Asn Asn He Met Phe Lys Leu Asp Gly Cys Arg Aεp Thr His Arg Tyr 195 200 205 ATC TGG ATC AAA TAC TTC AAT CTG TTC GAC AAA GAA CTG AAC GAA AAA 672 He Trp He Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys 210 215 220
GAA ATC AAA GAC CTG TAC GAC AAC CAG TCC AAT TCT GGT ATC CTG AAA 720 Glu He Lys Asp Leu Tyr Asp Asn Gin Ser Asn Ser Gly He Leu Lyε 225 ' 230 235 240
GAC TTC TGG GGT GAC TAC CTG CAG TAC GAC AAA CCG TAC TAC ATG CTG 768 Asp Phe Trp Gly Asp Tyr Leu Gin Tyr Asp Lys Pro Tyr Tyr Met Leu 245 250 255
AAT CTG TAC GAT CCG AAC AAA TAC GTT GAC GTC AAC AAT GTA GGT ATC 816 Aεn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly He 260 265 270 CGC GGT TAC ATG TAC CTG AAA GGT CCG CGT GGT TCT GTT ATG ACT ACC 864
Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr
275 280 285 AAC ATC TAC CTG AAC TCT TCC CTG TAC CGT GGT ACC AAA TTC ATC ATC 912
Asn He Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe He He
290 295 300
AAG AAA TAC GCG TCT GGT AAC AAG GAC AAT ATC GTT CGC AAC AAT GAT 960 Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn He Val Arg Asn Asn Asp
305 ' " 310 315 320
CGT GTA TAC ATC AAT GTT GTA GTT AAG AAC AAA GAA TAC CGT CTG GCT 1008
Arg Val Tyr He Asn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala 325 330 335
ACC AAT GCT TCT CAG GCT GGT GTA GAA AAG ATC TTG TCT GCT CTG GAA 1056
Thr Asn Ala Ser Gin Ala Gly Val Glu Lys He Leu Ser Ala Leu Glu
340 345 350
ATC CCG GAC GTT GGT AAT CTG TCT CAG GTA GTT GTA ATG AAA TCC AAG 1104
He Pro Asp Val Gly Asn Leu Ser Gin Val Val Val Met Lys Ser Lys
355 360 365 AAC GAC CAG GGT ATC ACT AAC AAA TGC AAA ATG AAT CTG CAG GAC AAC 1152
Asn Asp Gin Gly He Thr Asn Lvs Cys Lyε Met Asn Leu Gin Asp Asn
370 375 ' ' 380
AAT GGT AAC GAT ATC GGT TTC ATC GGT TTC CAC CAG TTC AAC AAT ATC 1200 Asn Gly Asn Asp He Gly Phe He Gly Phe His Gin Phe Asn Asn He
385 390 395 400
GCT AAA CTG GTT GCT TCC AAC TGG TAC AAT CGT CAG ATC GAA CGT TCC 1248
Ala Lys Leu Val Ala Ser Asn Trp Tyr Aεn Arg Gin He Glu Arg Ser 405 410 415
TCT CGC ACT CTG GGT TGC TCT TGG GAG TTC ATC CCG GTT GAT GAC GGT 1296
Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe He Pro Val Asp Aεp Gly
420 425 430
TGG GGT GAA CGT CCG CTG TAACCCGGGA AAGCTT 1330
Trp Gly Glu Arg Pro Leu 435 (2) INFORMATION FOR SEQ ID NO: 23:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 23 :
Met Ala Arg Leu Leu Ser Thr Phe Thr Glu Tyr He Lys Asn He He
1 5 10 15
Asn Thr Ser He Leu Asn Leu Arg Tyr Glu Ser Asn His Leu He Aεp 20 25 30
Leu Ser Arg Tyr Ala Ser Lys He Asn He Gly Ser Lys Val Asn Phe
35 40 45 Asp Pro He Asp Lys Asn Gin He Gin Leu Phe Asn Leu Glu Ser Ser
50 55 60
Lys He Glu Val He Leu Lys Asn Ala He Val Tyr Asn Ser Met Tyr
65 70 ' 75 80 Glu Asn Phe Ser Thr Ser Phe Trp He Arg He Pro Lys Tyr Phe Asn 85 90 95
Ser He Ser Leu Asn Asn Glu Tyr Thr He He Asn Cys Met Glu Asn 100 105 110
Asn Ser Gly Trp Lys Val Ser Leu Asn Tyr Gly Glu He He Trp Thr 115 120 125 Leu Gin Asp Thr Gin Glu He Lys Gin Arg Val Val Phe Lys Tyr Ser 130 135 140
Gin Met He Asn He Ser Asp Tyr He Asn Arg Trp He Phe Val Thr 145 150 155 160
He Thr Asn Asn Arg Leu Asn Asn Ser Lys He Tyr He Asn Gly Arg 165 170 175
Leu He Asp Gin Lys Pro He Ser Asn Leu Gly Asn He His Ala Ser 180 185 190
Asn Asn He Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr 195 200 205 He Trp He Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys 210 215 220
Glu He Lys Asp Leu Tyr Asp Asn Gin Ser Asn Ser Gly He Leu Lys 225 230 235 240
Asp Phe Trp Gly Asp Tyr Leu Gin Tyr Asp Lys Pro Tyr Tyr Met Leu 245 250 255
Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val Gly He 260 265 270
Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val Met Thr Thr 275 280 285 Asn He Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr Lys Phe He He 290 295 300
Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn He Val Arg Asn Asn Asp
305 ' 310 315 320
Arg Val Tyr He Aεn Val Val Val Lys Asn Lys Glu Tyr Arg Leu Ala
325 330 335
Thr Asn Ala Ser Gin Ala Gly Val Glu Lys He Leu Ser Ala Leu Glu 340 345 350
He Pro Asp Val Gly Asn Leu Ser Gin Val Val Val Met Lys Ser Lyε 355 360 365
Asn Asp Gin Gly He Thr Asn Lys Cys Lys Met Asn Leu Gin Aεp Asn
370 J 375 380
Asn Gly Asn Asp He Gly Phe He Gly Phe His Gin Phe Aεn Asn He
385 390 395 400
Ala Lys Leu Val Ala Ser Asn Trp Tyr Asn Arg Gin He Glu Arg Ser
405 410 415
Ser Arg Thr Leu Gly Cys Ser Trp Glu Phe He Pro Val Asp Asp Gly 420 ' 425 430
Trp Gly Glu Arg Pro Leu 435 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 ammo acids
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown (D) TOPOLOGY: linear
(il) MOLECULE TYPE: prote
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24.
Met Gly His His His His His His His His His His Ser Ser Gly His
1 5 10 15
Figure imgf000267_0001
(2) INFORMATION FOR SEQ ID NO: 25:
(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1402 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1386
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATG GGC CAT CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT 48 Met Gly His His His His His His His Hiε His His Ser Ser Gly His 1 5 10 15
ATC GAA GGT CGT CAT ATG GCT AGC ATG GCT CGT CTG CTG TCT ACC TTC 96
He Glu Gly Arg His Met Ala Ser Met Ala Arg Leu Leu Ser Thr Phe
20 25 30
ACT GAA TAC ATC AAG AAC ATC ATC AAT ACC TCC ATC CTG AAC CTG CGC 144
Thr Glu Tyr He Lys Asn He He Asn Thr Ser He Leu Asn Leu Arg
35 40 45 TAC GAA TCC AAT CAC CTG ATC GAC CTG TCT CGC TAC GCT TCC AAA ATC 192
Tyr Glu Ser Asn His Leu He Asp Leu Ser Arg Tyr Ala Ser Lys He 50 55 60
AAC ATC GGT TCT AAA GTT AAC TTC GAT CCG ATC GAC AAG AAT CAG ATC 240 Asn He Gly Ser Lys Val Asn Phe Asp Pro He Asp Lys Asn Gin He 65 70 75 80
CAG CTG TTC AAT CTG GAA TCT TCC AAA ATC GAA GTT ATC CTG AAG AAT 288
Gin Leu Phe Asn Leu Glu Ser Ser Lys He Glu Val He Leu Lys Asn 85 90 95
GCT ATC GTA TAC AAC TCT ATG TAC GAA AAC TTC TCC ACC TCC TTC TGG 336
Ala He Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp
100 ' 105 110
ATC CGT ATC CCG AAA TAC TTC AAC TCC ATC TCT CTG AAC AAT GAA TAC 384 He Arg He Pro Lys Tyr Phe Asn Ser He Ser Leu Asn Asn Glu Tvr 115 120 125 ACC ATC ATC AAC TGC ATG GAA AAC AAT TCT GGT TGG AAA GTA TCT CTG 432 Thr He He Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu 130 135 140
AAC TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACT CAG GAA ATC AAA 480 Asn Tyr Gly Glu He He Trp Thr Leu Gin Asp Thr Gin Glu He Lys 145 150 155 160
CAG CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC 528 Gin Arg Val Val Phe Lys Tyr Ser Gin Met He Asn He Ser Asp Tyr 165 170 175
ATC AAT CGC TGG ATC TTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC 576 He Asn Arg Trp He Phe Val Thr He Thr Asn Asn Arg Leu Asn Asn 180 185 190
TCC AAA ATC TAC ATC AAC GGC CGT CTG ATC GAC CAG AAA CCG ATC TCC 624 Ser Lys He Tyr He Asn Gly Arg Leu He Asp Gin Lys Pro He Ser 195 200 205
AAT CTG GGT AAC ATC CAC GCT TCT AAT AAC ATC ATG TTC AAA CTG GAC 672 Asn Leu Gly Asn He His Ala Ser Asn Asn He Met Phe Lys Leu Asp 210 215 220
GGT TGT CGT GAC ACT CAC CGC TAC ATC TGG ATC AAA TAC TTC AAT CTG 720 Gly Cys Arg Asp Thr His Arg Tyr He Trp He Lys Tyr Phe Asn Leu 225 230 235 240
TTC GAC AAA GAA CTG AAC GAA AAA GAA ATC AAA GAC CTG TAC GAC AAC 768 Phe Asp Lys Glu Leu Asn Glu Lys Glu He Lys Asp Leu Tyr Asp Asn 245 250 255
CAG TCC AAT TCT GGT ATC CTG AAA GAC TTC TGG GGT GAC TAC CTG CAG 816 Gin Ser Asn Ser Gly He Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gin 260 265 270
TAC GAC AAA CCG TAC TAC ATG CTG AAT CTG TAC GAT CCG AAC AAA TAC 864 Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr 275 280 285 GTT GAC GTC AAC AAT GTA GGT ATC CGC GGT TAC ATG TAC CTG AAA GGT 912 Val Asp Val Asn Aεn Val Gly He Arg Gly Tyr Met Tyr Leu Lys Gly 290 295 300
CCG CGT GGT TCT GTT ATG ACT ACC AAC ATC TAC CTG AAC TCT TCC CTG 960 Pro Arg Gly Ser Val Met Thr Thr Asn He Tyr Leu Asn Ser Ser Leu 305 310 315 320
TAC CGT GGT ACC AAA TTC ATC ATC AAG AAA TAC GCG TCT GGT AAC AAG 1008 Tyr Arg Gly Thr Lys Phe He He Lys Lys Tyr Ala Ser Gly Asn Lys 325 ' 33θ ' 335 '
GAC AAT ATC GTT CGC AAC AAT GAT CGT GTA TAC ATC AAT GTT GTA GTT 1056
Asp Asn He Val Arg Asn Asn Asp Arg Val Tyr He Asn Val Val Val
340 " 345 350
AAG AAC AAA GAA TAC CGT CTG GCT ACC AAT GCT TCT CAG GCT GGT GTA 1104
Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala Gly Val 355 360 365 GAA AAG ATC TTG TCT GCT CTG GAA ATC CCG GAC GTT GGT AAT CTG TCT 1152 Glu Lyε He Leu Ser Ala Leu Glu He Pro Asp Val Gly Asn Leu Ser 370 375 380
CAG GTA GTT GTA ATG AAA TCC AAG AAC GAC CAG GGT ATC ACT AAC AAA 1200 Gin Val Val Val Met Lys Ser Lys Asn Asp Gin Gly He Thr Asn Lys 385 - 390 395 ' 400
TGC AAA ATG AAT CTG CAG GAC AAC AAT GGT AAC GAT ATC GGT TTC ATC 1248 Cys Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp He Gly Phe He 405 410 415 GGT TTC CAC CAG TTC AAC AAT ATC GCT AAA CTG GTT GCT TCC AAC TGG 1296 Gly Phe His Gin Phe Asn Asn He Ala Lys Leu Val Ala Ser Asn Trp 420 425 430 TAC AAT CGT CAG ATC GAA CGT TCC TCT CGC ACT CTG GGT TGC TCT TGG 1344 Tyr Asn Arg Gin He Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp 435 440 " 445
GAG TTC ATC' CCG GTT GAT GAC GGT TGG GGT GAA CGT CCG CTG 1386 Glu Phe He Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 450 455 460
TAACCCGGGA AAGCTT 1402 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 26 :
Met Gly His His His His His His His His His His Ser Ser Gly His 1 10 15
He Glu Gly Arg His Met Ala Ser Met Ala Arg Leu Leu Ser Thr Phe ' 20 25 30
Thr Glu Tyr He Lys Asn He He Asn Thr Ser He Leu Asn Leu Arg 35 40 45 Tyr Glu Ser Asn His Leu He Asp Leu Ser Arg Tyr Ala Ser Lys lie
50 55 60
Asn He Gly Ser Lys Val Asn Phe Asp Pro He Asp Lys Asn Gin He 65 70 75 80
Gin Leu Phe Asn Leu Glu Ser Ser Lys He Glu Val He Leu Lys Asn 85 90 95
Ala He Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp 100 105 110
He Arg He Pro Lys Tyr Phe Asn Ser He Ser Leu Asn Asn Glu Tyr 115 120 125
Thr Ile He Asn Cys Met Glu Asn Asn Ser Gly Trp Lys V l Ser Leu 130 135 140
Asn Tyr Gly Glu He He Trp Thr Leu Gin Asp Thr Gin Glu He Lys 145 150 155 160
Gin Arg Val Val Phe Lys Tyr Ser Gin Met He Asn He Ser Asp Tyr 165 170 175
He Asn Arg Trp He Phe Val Thr He Thr Asn Asn Arg Leu Asn Asn 180 185 190
Figure imgf000270_0001
Asp Asn He Val Arg Aεn Aεn Asp Arg Val Tyr He Asn Val Val Val 340 345 350
Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala Gly Val 355 360 365
Glu Lys He Leu Ser Ala Leu Glu He Pro Asp Val Gly Asn Leu Ser 370 375 380
Gin Val Val Val Met Lyε Ser Lys Asn Asp Gin Gly He Thr Asn Lys 385 390 395 400 " s Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp He Gly Phe He
405 410 415
Gly Phe Hrs Gin Phe Asn Asn He Ala Lys Leu Val Ala Ser Asn Trp 420 425 430
Tyr Asn Arg Gin He Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp 435 440 445
Glu Phe He Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 450 455 460
(2) INFORMATION FOR SEQ ID NO .27
(l) SEQUENCE CHARACTERISTICS. (A) LENGTH: 3891 base pairε
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY, linear Ui) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3888
(XI) SEQUENCE DESCRIPTION: SEQ ID NO:27:
ATG CAA TTT GTT AAT AAA CAA TTT AAT TAT AAA GAT CCT GTA AAT GGT 48
Met Gin Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly
1 5 10 15
GTT GAT ATT GCT TAT ATA AAA ATT CCA AAT GTA GGA CAA ATG CAA CCA 96
Val Asp He Ala Tyr He Lys He Pro Asn Val Gly Gin Met Gin Pro 20 25 30 GTA AAA GCT TTT AAA ATT CAT AAT AAA ATA TGG GTT ATT CCA GAA AGA 144 Val Lys Ala Phe Lys He His Asn Lys He Trp Val He Pro Glu Arg 35 40 45
GAT ACA TTT ACA AAT CCT GAA GAA GGA GAT TTA AAT CCA CCA CCA GAA 192 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
GCA AAA CAA GTT CCA GTT TCA TAT TAT GAT TCA ACA TAT TTA AGT ACA 240 Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
GAT AAT GAA AAA GAT AAT TAT TTA AAG GGA GTT ACA AAA TTA TTT GAG 288
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
AGA ATT TAT TCA ACT GAT CTT GGA AGA ATG TTG TTA ACA TCA ATA GTA 336
Arg He Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser He Val 100 105 110 AGG GGA ATA CCA TTT TGG GGT GGA AGT ACA ATA GAT ACA GAA TTA AAA 384 Arg Gly He Pro Phe Trp Gly Gly Ser Thr He Asp Thr Glu Leu Lys 115 ' 120 125
GTT ATT GAT ACT AAT TGT ATT AAT GTG ATA CAA CCA GAT GGT AGT TAT 432 Val He Asp Thr Asn Cys He Asn Val He Gin Pro Asp Gly Ser Tyr 130 135 140
AGA TCA GAA GAA CTT AAT CTA GTA ATA ATA GGA CCC TCA GCT GAT ATT 480 Arg Ser Glu Glu Leu Asn Leu Val He He Gly Pro Ser Ala Asp He 145 150 155 160
ATA CAG TTT GAA TGT AAA AGC TTT GGA CAT GAA GTT TTG AAT CTT ACG 528
He Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr
165 170 175
CGA AAT GGT TAT GGC TCT ACT CAA TAC ATT AGA TTT AGC CCA GAT TTT 576 Arg Asn Gly Tyr Gly Ser Thr Gin Tyr He Arg Phe Ser Pro Asp Phe 180 185 190 ACA TTT GGT TTT GAG GAG TCA CTT GAA GTT GAT ACA AAT CCT CTT TTA 624 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
GGT GCA GGC AAA TTT GCT ACA GAT CCA GCA GTA ACA TTA GCA CAT GAA 672 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 ' 215 220
CTT ATA CAT GCT GGA CAT AGA TTA TAT GGA ATA GCA ATT AAT CCA AAT 720 Leu He His Ala Gly His Arg Leu Tyr Gly He Ala He Asn Pro Asn 225 230 235 240
AGG GTT TTT AAA GTA AAT ACT AAT GCC TAT TAT GAA ATG AGT GGG TTA 768 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 GAA GTA AGC TTT GAG GAA CTT AGA ACA TTT GGG GGA CAT GAT GCA AAG 816 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 TTT ATA GAT AGT TTA CAG GAA AAC GAA TTT CGT CTA TAT TAT TAT AAT 864 Phe He Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
AAG TTT AAA GAT ATA GCA AGT ACA CTT AAT AAA GCT AAA TCA ATA GTA 912 Lys Phe Lys Asp He Ala Ser Thr Leu Asn Lys Ala Lys Ser He Val 290 295 300
GGT ACT ACT GCT TCA TTA CAG TAT ATG AAA AAT GTT TTT AAA GAG AAA 960 Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 15 320
TAT CTC CTA TCT GAA GAT ACA TCT GGA AAA TTT TCG GTA GAT AAA TTA 1008
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu
325 330 335
AAA TTT GAT AAG TTA TAC AAA ATG TTA ACA GAG ATT TAC ACA GAG GAT 1056
Lys Phe Asp Lyε Leu Tyr Lys Met Leu Thr Glu He Tyr Thr Glu Asp 340 345 350 AAT TTT GTT AAG TTT TTT AAA GTA CTT AAC AGA AAA ACA TAT TTG AAT 1104 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365
TTT GAT AAA GCC GTA TTT AAG ATA AAT ATA GTA CCT AAG GTA AAT TAC 1152 Phe Asp Lys Ala Val Phe Lys He Asn He Val Pro Lys Val Asn Tyr 370 375 380
ACA ATA TAT GAT GGA TTT AAT TTA AGA AAT ACA AAT TTA GCA GCA AAC 1200 Thr lie Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400
TTT AAT GGT CAA AAT ACA GAA ATT AAT AAT ATG AAT TTT ACT AAA CTA 1248
Phe Asn Gly Gin Asn Thr Glu He Asn Asn Met Asn Phe Thr Lys Leu
405 410 415
AAA AAT TTT ACT GGA TTG TTT GAA TTT TAT AAG TTG CTA TGT GTA AGA 1296
Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 * ' 430 GGG ATA ATA ACT TCT AAA ACT AAA TCA TTA GAT AAA GGA TAC AAT AAG 1344 Gly He He Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445
GCA TTA AAT GAT TTA TGT ATC AAA GTT AAT AAT TGG GAC TTG TTT TTT 1392 Ala Leu Asn Asp Leu Cys He Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460
AGT CCT TCA GAA GAT AAT TTT ACT AAT GAT CTA AAT AAA GGA GAA GAA 1440 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480
ATT ACA TCT GAT ACT AAT ATA GAA GCA GCA GAA GAA AAT ATT AGT TTA 1488 He Thr Ser Asp Thr Asn He Glu Ala Ala Glu Glu Asn He Ser Leu 485 490 495
GAT TTA ATA CAA CAA TAT TAT TTA ACC TTT AAT TTT GAT AAT GAA CCT 1536 Asp Leu He Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 GAA AAT ATT TCA ATA GAA AAT CTT TCA AGT GAC ATT ATA GGC CAA TTA 1584 Glu Asn He Ser He Glu Asn Leu Ser Ser Asp He He Gly Gin Leu 515 520 525
GAA CTT ATG CCT AAT ATA GAA AGA TTT CCT AAT GGA AAA AAG TAT GAG 1632 Glu Leu Met Pro Asn He Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540
TTA GAT AAA TAT ACT ATG TTC CAT TAT CTT CGT GCT CAA GAA TTT GAA 1680 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu 545 550 555 560
CAT GGT AAA TCT AGG ATT GCT TTA ACA AAT TCT GTT AAC GAA GCA TTA 1728
His Gly Lys Ser Arg He Ala Leu Thr Asn Ser Val Asn Glu Ala Leu
565 570 575
TTA AAT CCT AGT CGT GTT TAT ACA TTT TTT TCT TCA GAC TAT GTA AAG 1776
Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590 AAA GTT AAT AAA GCT ACG GAG GCA GCT ATG TTT TTA GGC TGG GTA GAA 1824 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605
CAA TTA GTA TAT GAT TTT ACC GAT GAA ACT AGC GAA GTA AGT ACT ACG 1872 Gin Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620
GAT AAA ATT GCG GAT ATA ACT ATA ATT ATT CCA TAT ATA GGA CCT GCT 1920 Asp Lys He Ala Asp He Thr He He He Pro Tyr He Gly Pro Ala 625 630 635 640
TTA AAT ATA GGT AAT ATG TTA TAT AAA GAT GAT TTT GTA GGT GCT TTA 1968
Leu Asn He Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
645 650 655
ATA TTT TCA GGA GCT GTT ATT CTG TTA GAA TTT ATA CCA GAG ATT GCA 2016
He Phe Ser Gly Ala Val He Leu Leu Glu Phe He Pro Glu He Ala
660 665 670 ATA CCT GTA TTA GGT ACT TTT GCA CTT GTA TCA TAT ATT GCG AAT AAG 2064 He Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr He Ala Asn Lys 675 680 685
GTT CTA ACC GTT CAA ACA ATA GAT AAT GCT TTA AGT AAA AGA AAT GAA 2112 Val Leu Thr Val Gin Thr He Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700
AAA TGG GAT GAG GTC TAT AAA TAT ATA GTA ACA AAT TGG TTA GCA AAG 2160 Lys Trp Asp Glu Val Tyr Lys Tyr He Val Thr Asn Trp Leu Ala Lys 705 710 " 715 "20
GTT AAT ACA CAG ATT GAT CTA ATA AGA AAA AAA ATG AAA GAA GCT TTA 2208
Val Asn Thr Gin He Asp Leu He Arg Lys Lys Met Lys Glu Ala Leu 725 730 735
GAA AAT CAA GCA GAA GCA ACA AAG GCT ATA ATA AAC TAT CAG TAT AAT 2256
Glu Asn Gin Ala Glu Ala Thr Lyε Ala He He Asn Tyr Gin Tyr Asn 740 745 750 CAA TAT ACT GAG GAA GAG AAA AAT AAT ATT AAT TTT AAT ATT GAT GAT 2304 Gin Tyr Thr Glu Glu Glu Lys Asn Asn He Asn Phe Asn He Asp Asp 755 760 765
TTA AGT TCG AAA CTT AAT GAG TCT ATA AAT AAA GCT ATG ATT AAT ATA 2352 Leu Ser Ser Lys Leu Asn Glu Ser He Asn Lys Ala Met He Asn He 770 " 775 780
AAT AAA TTT TTG AAT CAA TGC TCT GTT TCA TAT TTA ATG AAT TCT ATG 2400 Asn Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800
ATC CCT TAT GGT GTT AAA CGG TTA GAA GAT TTT GAT GCT AGT CTT AAA 2448 He Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815 GAT GCA TTA TTA AAG TAT ATA TAT GAT AAT AGA GGA ACT TTA ATT GGT 2496 Asp Ala Leu Leu Lys Tyr He Tyr Asp Asn Arg Gly Thr Leu He Gly 820 825 830 CAA GTA GAT AGA TTA AAA GAT AAA GTT AAT AAT ACA CTT AGT ACA GAT 2544 Gin Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845
ATA CCT TTT CAG CTT TCC AAA TAC GTA GAT AAT CAA AGA TTA TTA TCT 2592 He Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg Leu Leu Ser 850 855 860
ACA TTT ACT GAA TAT ATT AAG AAT ATT ATT AAT ACT TCT ATA TTG AAT 2640 Thr Phe Thr Glu Tyr He Lys Asn He He Asn Thr Ser He Leu Asn 865 870 875 880
TTA AGA TAT GAA AGT AAT CAT TTA ATA GAC TTA TCT AGG TAT GCA TCA 2688
Leu Arg Tyr Glu Ser Asn His Leu He Asp Leu Ser Arg Tyr Ala Ser
885 890 895
AAA ATA AAT ATT GGT AGT AAA GTA AAT TTT GAT CCA ATA GAT AAA AAT 2736
Lys He Asn He Gly Ser Lys Val Asn Phe Asp Pro He Asp Lys Asn
900 905 910 CAA ATT CAA TTA TTT AAT TTA GAA AGT AGT AAA ATT GAG GTA ATT TTA 2784 Gin He Gin Leu Phe Asn Leu Glu Ser Ser Lys He Glu Val He Leu 915 920 925
AAA AAT GCT ATT GTA TAT AAT AGT ATG TAT GAA AAT TTT AGT ACT AGC 2832 Lys Asn Ala He Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940
TTT TGG ATA AGA ATT CCT AAG TAT TTT AAC AGT ATA AGT CTA AAT AAT 2880 Phe Trp He Arg He Pro Lys Tyr Phe Asn Ser He Ser Leu Asn Asn 945 950 955 960
GAA TAT ACA ATA ATA AAT TGT ATG GAA AAT AAT TCA GGA TGG AAA GTA 2928
Glu Tyr Thr He He Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975
TCA CTT AAT TAT GGT GAA ATA ATC TGG ACT TTA CAG GAT ACT CAG GAA 2976
Ser Leu Asn Tyr Gly Glu He He Trp Thr Leu Gin Asp Thr Gin Glu 980 985 990 ATA AAA CAA AGA GTA GTT TTT AAA TAC AGT CAA ATG ATT AAT ATA TCA 3024 He Lys Gin Arg Val Val Phe Lys Tyr Ser Gin Met He Asn He Ser 995 1000 1005
GAT TAT ATA AAC AGA TGG ATT TTT GTA ACT ATC ACT AAT AAT AGA TTA 3072 Asp Tyr He Asn Arg Trp He Phe Val Thr He Thr Asn Asn Arg Leu 1010 1015 1020
AAT AAC TCT AAA ATT TAT ATA AAT GGA AGA TTA ATA GAT CAA AAA CCA 3120 Asn Asn Ser Lys He Tyr He Asn Gly Arg Leu He Asp Gin Lys Pro 1025 1030 1035 1040
ATT TCA AAT TTA GGT AAT ATT CAT GCT AGT AAT AAT ATA ATG TTT AAA 3168
He Ser Asn Leu Gly Asn He His Ala Ser Asn Asn He Met Phe Lys 1045 1050 1055
TTA GAT GGT TGT AGA GAT ACA CAT AGA TAT ATT TGG ATA AAA TAT TTT 3216
Leu Asp Gly Cys Arg Asp Thr His Arg Tyr He Trp He Lys Tyr Phe 1060 1065 1070 AAT CTT TTT GAT AAG GAA TTA AAT GAA AAA GAA ATC AAA GAT TTA TAT 3264 Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu He Lys Asp Leu Tyr 1075 ' 1080 1085
GAT AAT CAA TCA AAT TCA GGT ATT TTA AAA GAC TTT TGG GGT GAT TAT 3312 Asp Aεn Gin Ser Asn Ser Gly He Leu Lyε Aεp Phe Trp Gly Aεp Tyr 1090 1095 1100
TTA CAA TAT GAT AAA CCA TAC TAT ATG TTA AAT TTA TAT GAT CCA AAT 3360 Leu Gin Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn 1105 1110 1115 1120
AAA TAT GTC GAT GTA AAT AAT GTA GGT ATT AGA GGT TAT ATG TAT CTT 3408 Lys Tyr Val Asp Val Asn Asn Val Gly He Arg Gly Tyr Met Tyr Leu 1125 1130 1135
AAA GGG CCT AGA GGT AGC GTA ATG ACT ACA AAC ATT TAT TTA AAT TCA 3456 Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn He Tyr Leu Asn Ser 1140 1145 1150
AGT TTG TAT AGG GGG ACA AAA TTT ATT ATA AAA AAA TAT GCT TCT GGA 3504 Ser Leu Tyr Arg Gly Thr Lys Phe He He Lys Lys Tyr Ala Ser Gly 1155 1160 1165
AAT AAA GAT AAT ATT GTT AGA AAT AAT GAT CGT GTA TAT ATT AAT GTA 3552 Asn Lys Asp Asn He Val Arg Asn Asn Asp Arg Val Tyr He Asn Val 1170 1175 1180
GTA GTT AAA AAT AAA GAA TAT AGG TTA GCT ACT AAT GCA TCA CAG GCA 3600 Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala 1185 1190 1195 1200
GGC GTA GAA AAA ATA CTA AGT GCA TTA GAA ATA CCT GAT GTA GGA AAT 3648 Gly Val Glu Lys He Leu Ser Ala Leu Glu He Pro Asp Val Gly Asn 1205 1210 1215
CTA AGT CAA GTA GTA GTA ATG AAG TCA AAA AAT GAT CAA GGA ATA ACA 3696 Leu Ser Gin Val Val Val Met Lys Ser Lys Asn Asp Gin Gly He Thr 1220 1225 1230 AAT AAA TGC AAA ATG AAT TTA CAA GAT AAT AAT GGG AAT GAT ATA GGC 3744 Asn Lys Cys Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp He Gly 1235 1240 1245
TTT ATA GGA TTT CAT CAG TTT AAT AAT ATA GCT AAA CTA GTA GCA AGT 3792 Phe He Gly Phe His Gin Phe Asn Asn He Ala Lys Leu Val Ala Ser 1250 1255 1260
AAT TGG TAT AAT AGA CAA ATA GAA AGA TCT AGT AGG ACT TTG GGT TGC 3840 Asn Trp Tyr Asn Arg Gin He Glu Arg Ser Ser Arg Thr Leu Gly Cyε 1265 1270 1275 1280
TCA TGG GAA TTT ATT CCT GTA GAT GAT GGA TGG GGA GAA AGG CCA CTG 3888 Ser Trp Glu Phe He Pro Val Asp Aεp Gly Trp Gly Glu Arg Pro Leu 1285 1290 1295
TAA 3891
(2) INFORMATION FOR SEQ ID NO: 28: U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1296 ammo acids
(B) TYPE: am o acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Met Gin Phe Val Asn Lys Gin Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 5 10 15
Val Asp He Ala Tyr He Lys He Pro Asn Val Gly Gin Met Gin Pro 20 25 30 Val Lys Ala Phe Lys He His Asn Lys He Trp Val He Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60
Ala Lys Gin Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80
Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95
Arg He Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser He Val 100 105 110
Arg Gly He Pro Phe Trp Gly Gly Ser Thr He Asp Thr Glu Leu Lys 115 120 * 125
Val He Asp Thr Asn Cys He Asn Val He Gin Pro Asp Gly Ser Tyr 130 135 140
Arg Ser Glu Glu Leu Asn Leu Val He He Gly Pro Ser Ala Asp He 145 150 155 160
He Gin Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gin Tyr He Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205
Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu
210 ' 215 220
Leu He His Ala Gly His Arg Leu Tyr Gly He Ala He Asn Pro Asn 225 230 235 240
Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255
Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe He Asp Ser Leu Gin Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285
Lys Phe Lys Asp He Ala Ser Thr Leu Asn Lys Ala Lys Ser He Val 290 295 300
Gly Thr Thr Ala Ser Leu Gin Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320
Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu 325 330 335
Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu He Tyr Thr Glu Asp
340 345 350
Asn Phe Val Lys Phe Phe Ly: V l Leu Asn Arg Lys Thr Tyr Leu Asn 3S5 360 365
Phe Asp Lys Ala Val Phe Lys He Asn He Val Pro Lys Val Aεn Tyr
370 375 380
Thr He Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn
385 390 395 400
Phe Asn Gly Gin Asn Thr Glu He Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430
Gly He He Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445
Ala Leu Asn Asp Leu Cys He Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460
Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480
He Thr Ser Asp Thr Asn He Glu Ala Ala Glu Glu Asn He Ser Leu 485 490 495
Asp Leu He Gin Gin Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510
Glu Asn He Ser He Glu Asn Leu Ser Ser Asp He He Gly Gin Leu 515 520 525
Glu Leu Met Pro Asn He Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540
Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gin Glu Phe Glu 545 550 555 560
His Gly Lys Ser Arg He Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575
Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590
Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605
Gin Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr
610 615 620
Asp Lys He Ala Asp He Thr He He He Pro Tyr He Gly Pro Ala
625 630 635 640
Leu Asn He Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
645 650 655
He Phe Ser Gly Ala Val He Leu Leu Glu Phe He Pro Glu He Ala
660 665 670
He Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr He Ala Asn Lyε 675 680 ' 685
Val Leu Thr Val Gin Thr He Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr He Val Thr Aεn Trp Leu Ala Lys 705 710 715 720
Val Asn Thr Gin He Asp Leu He Arg Lys Lys Met Lys Glu Ala Leu
725 730 735
Glu Asn Gin Ala Glu Ala Thr Lys Ala He He Asn Tyr Gin Tyr Asn
740 745 750
Gin Tyr Thr Glu Glu Glu Lys Asn Asn He Asn Phe Asn He Asp Asp 755 760 765
Leu Ser Ser Lys Leu Asn Glu Ser He Asn Lys Ala Met He Asn He 770 ' 775 780 Asn Lys Phe Leu Asn Gin Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800
He Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815
Asp Ala Leu Leu Lys Tyr He Tyr Asp Asn Arg Gly Thr Leu He Gly 820 ' 825 830
Gin Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845
He Pro Phe Gin Leu Ser Lys Tyr Val Asp Asn Gin Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr He Lys Asn He He Asn Thr Ser He Leu Asn 865 870 875 880
Leu Arg Tyr Glu Ser Asn His Leu He Asp Leu Ser Arg Tyr Ala Ser 885 890 895
Lyε He Aεn He Gly Ser Lys Val Asn Phe Asp Pro He Asp Lys Asn 900 905 910
Gin He Gin Leu Phe Asn Leu Glu Ser Ser Lyε He Glu Val He Leu 915 920 925
Lys Asn Ala He Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940 Phe Trp He Arg He Pro Lyε Tyr Phe Asn Ser He Ser Leu Asn Asn 945 950 955 960
Glu Tyr Thr He He Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975
Ser Leu Asn Tyr Gly Glu He He Trp Thr Leu Gin Asp Thr Gin Glu 980 985 990
He Lys Gin Arg Val Val Phe Lys Tyr Ser Gin Met He Asn He Ser 995 1000 1005
Asp Tyr He Aεn Arg Trp He Phe Val Thr He Thr Aεn Asn Arg Leu 1010 ' 1015 1020 Asn Aεn Ser Lyε He Tyr He Asn Gly Arg Leu He Asp Gin Lys Pro 1025 ' 1030 1035 1040
He Ser Asn Leu Gly Asn He His Ala Ser Asn Asn He Met Phe Lys 1045 1050 1055
Leu Asp Gly Cys Arg Asp Thr His Arg Tyr He Trp He Lys Tyr Phe 1060 1065 1070
Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu He Lys Asp Leu Tyr 1075 1080 1085
Asp Asn Gin Ser Asn Ser Gly He Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 Leu Gin Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn 1105 " 1110 ' 1115 1120
Lys Tyr Val Asp Val Asn Asn Val Gly He Arg Gly Tyr Met Tyr Leu 1125 1130 1135
Lys Gly Pro Arg Gly Ser Val Met Thr Thr Asn He Tyr Leu Asn Ser 1140 1145 1150
Ser Leu Tyr Arg Gly Thr Lys Phe He He Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Lys Asp Asn He Val Arg Asn Asn Asp Arg Val Tyr He Asn Val 1170 * 1175 1180
Val Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala 1185 1190 1195 1200
Gly Val Glu Lys He Leu Ser Ala Leu Glu He Pro Asp Val Gly Asn 1205 1210 1215
Leu Ser Gin Val Val Val Met Lys Ser Lys Asn Asp Gin Gly He Thr 1220 1225 1230
Asn Lys Cys Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp He Gly 1235 1240 1245
Phe He Gl/ Phe His Gin Phe Aεn Asn He Ala Lys Leu Val Ala Ser 1250 1255 1260
Asn Trp Tyr Asn Arg Gin He Glu Arg Ser Ser Arg Thr Leu Gly Cys 1265 1270 1275 1280
Ser Trp Glu Phe He Pro Val Asp Asp Gly Trp Glv Glu Arg Pro Leu 1285 1290 1295
(2) INFORMATION FOR SEQ ID NO 29
(r) SEQUENCE CHARACTERISTICS
(A) LENGTH 30 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single 'D) TOPOLOGY linear
!n) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "DNA"
( .i) SEQUENCE DESCRIPTION SEQ ID NO 29 CGCCATGGCT AGATTATTAT CTACATTTAC 30 (2) INFORMATION FOR SEQ ID NO 30
U) SEQUENCE CHARACTERISTICS
(A) LENGTH 26 base pairs
(B) TYPE nucleic acid <C) STRANDEDNESS single
(D) TOPOLOGY linear ii) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "DNA"
(xi ) SEQUENCE DESCRIPTION SEQ ID NO 30 GCAAGCTTCT TGACAGACTC ATGTAG 26 (2) INFORMATION FOR SEQ ID NO 31
(l) SEQUENCE CHARACTERISTICS
(A) LENGTH 1546 base pairs
(B) TYPE nucleic acid (C) STRANDEDNESS double
"(D) TOPOLOGY linear
(n) MOLECULE TYPE DNA (genomic) fκi) SEQUENCE DESCRIPTION SEQ ID NO 31
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACCATG GGCCATCATC 120 ATCATCATCA TCATCATCAT CACAGCAGCG GCCATATCGA AGGTCGTCAT ATGGCTAGCA 180
TGGCTAGATT ATTATCTACA TTTACTGAAT ATATTAAGAA TATTATTAAT ACTTCTATAT 240
TGAATTTAAG ATATGAAAGT AATCATTTAA TAGACTTATC TAGGTATGCA TCAAAAATAA 300
ATATTGGTAG TAAAGTAAAT TTTGATCCAA TAGATAAAAA TCAAATTCAA TTATTTAATT 360
TAGAAAGTAG TAAAATTGAG GTAATTTTAA AAAATGCTAT TGTATATAAT AGTATGTATG 420
AAAATTTTAG TACTAGCTTT TGGATAAGAA TTCCTAAGTA TTTTAACAGT ATAAGTCTAA 480
ATAATGAATA TACAATAATA AATTGTATGG AAAATAATTC AGGATGGAAA GTATCACTTA 540
ATTATGGTGA AATAATCTGG ACTTTACAGG ATACTCAGGA AATAAAACAA AGAGTAGTTT 600
TTAAATACAG TCAAATGATT AATATATCAG ATTATATAAA CAGATGGATT TTTGTAACTA 660
TCACTAATAA TAGATTAAAT AACTCTAAAA TTTATATAAA TGGAAGATTA ATAGATCAAA 720
AACCAATTTC AAATTTAGGT AATATTCATG CTAGTAATAA TATAATGTTT AAATTAGATG 780
GTTGTAGAGA TACACATAGA TATATTTGGA TAAAATATTT TAATCTTTTT GATAAGGAAT 840
TAAATGAAAA AGAAATCAAA GATTTATATG ATAATCAATC AAATTCAGGT ATTTTAAAAG 900
ACTTTTGGGG TGATTATTTA CAATATGATA AACCATACTA TATGTTAAAT TTATATGATC 960
CAAATAAATA TGTCGATGTA AATAATGTAG GTATTAGAGG TTATATGTAT CTTAAAGGGC 1020
CTAGAGGTAG CGTAATGACT ACAAACATTT ATTTAAATTC AAGTTTGTAT AGGGGGACAA 1080
AATTTATTAT AAAAAAATAT GCTTCTGGAA ATAAAGATAA TATTGTTAGA AATAATGATC 1140 GTGTATATAT TAATGTAGTA GTTAAAAATA AAGAATATAG GTTAGCTACT AATGCATCAC 1200
AGGCAGGCGT AGAAAAAATA CTAAGTGCAT TAGAAATACC TGATGTAGGA AATCTAAGTC 1260
AAGTAGTAGT AATGAAGTCA AAAAATGATC AAGGAATAAC AAATAAATGC AAAATGAATT 1320
TACAAGATAA TAATGGGAAT GATATAGGCT TTATAGGATT TCATCAGTTT AATAATATAG 1380
CTAAACTAGT AGCAAGTAAT TGGTATAATA GACAAATAGA AAGATCTAGT AGGACTTTGG 1440 GTTGCTCATG GGAATTTATT CCTGTAGATG ATGGATGGGG AGAAAGGCCA CTGTAATTAA 1500
TCTCAAACTA CATGAGTCTG TCAAGAAGCT TGCGGCCGCA CTCGAG 1546
(2) INFORMATION FOR SEQ ID NO: 32:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: ammo acid
(C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant
(n) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
Met His His H s His His His Met Ala 1 5
INFORMATION FOR SEQ ID NO: 33:
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
TATGCATCAC CATCACCATC A 21
(2) INFORMATION FOR SEQ ID NO: 34
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
CATGTGATGG TGATGGTGAT GCA 23
■2) INFORMATION FOR SEQ ID NO: 35:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1351 base pairε
(B) TYPE: nucleic acid
(C) STRANDEDNESS- double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE, other nucleic acid (A) DESCRIPTION: /desc = "DNA"
Ux) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION- 1..1335
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 35 : ATG CAT CAC CAT CAC CAT CAC ATG GCT CGT CTG CTG TCT ACC TTC ACT 48
Met His His His His His His Met Ala Arg Leu Leu Ser Thr Phe Thr
1 10 15
GAA TAC ATC AAG AAC ATC ATC AAT ACC TCC ATC CTG AAC CTG CGC TAC 96 Glu Tyr He Lys Asn He He Asn Thr Ser He Leu Asn Leu Arg Tyr
20 25 30
GAA TCC AAT CAC CTG ATC GAC CTG TCT CGC TAC GCT TCC AAA ATC AAC 144 Glu Ser Asn His Leu He Asp Leu Ser Arg Tyr Ala Ser Lys He Asn 35 40 45
ATC GGT TCT AAA GTT AAC TTC GAT CCG ATC GAC AAG AAT CAG ATC CAG 192 He Gly Ser Lys Val Asn Phe Asp Pro He Asp Lys Asn Gin He Gin 50 55 60
CTG TTC AAT CTG GAA TCT TCC AAA ATC GAA GTT ATC CTG AAG AAT GCT 240 Leu Phe Asn Leu Glu Ser Ser Lys He Glu Val He Leu Lys Asn Ala 65 70 75 80 ATC GTA TAC AAC TCT ATG TAC GAA AAC TTC TCC ACC TCC TTC TGG ATC 288 He Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp He 85 90 95
CGT ATC CCG AAA TAC TTC AAC TCC ATC TCT CTG AAC AAT GAA TAC ACC 336 Arg He Pro Lys Tyr Phe Asn Ser He Ser Leu Asn Asn Glu Tyr Thr
100 105 110
ATC ATC AAC TGC ATG GAA AAC AAT TCT GGT TGG AAA GTA TCT CTG AAC 384 He He Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn 115 120 125 TAC GGT GAA ATC ATC TGG ACT CTG CAG GAC ACT CAG GAA ATC AAA CAG 432 Tyr Gly Glu He He Trp Thr Leu Gin Asp Thr Gin Glu He Lys Gin 130 135 140
CGT GTT GTA TTC AAA TAC TCT CAG ATG ATC AAC ATC TCT GAC TAC ATC 480 Arg Val Val Phe Lys Tyr Ser Gin Met He Asn He Ser Asp Tyr He 145 150 155 160
AAT CGC TGG ATC TTC GTT ACC ATC ACC AAC AAT CGT CTG AAT AAC TCC 528 Asn Arg Trp He Phe Val Thr He Thr Asn Asn Arg Leu Asn Asn Ser 165 170 175
AAA ATC TAC ATC AAC GGC CGT CTG ATC GAC CAG AAA CCG ATC TCC AAT 576 Lys He Tyr He Asn Gly Arg Leu He Asp Gin Lys Pro He Ser Asn 180 185 ' 190
CTG GGT AAC ATC CAC GCT TCT AAT AAC ATC ATG TTC AAA CTG GAC GGT 624 Leu Gly Asn He His Ala Ser Asn Asn He Met Phe Lys Leu Asp Gly 195 200 205
TGT CGT GAC ACT CAC CGC TAC ATC TGG ATC AAA TAC TTC AAT CTG TTC 672 Cys Arg Asp Thr His Arg Tyr He Trp He Lys Tyr Phe Asn Leu Phe 210 215 220 GAC AAA GAA CTG AAC GAA AAA GAA ATC AAA GAC CTG TAC GAC AAC CAG 720 Asp Lys Glu Leu Asn Glu Lys Glu He Lys Asp Leu Tyr Asp Asn Gin 225 230 ' 235 240
TCC AAT TCT GGT ATC CTG AAA GAC TTC TGG GGT GAC TAC CTG CAG TAC 768 Ser Aεn Ser Gly He Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gin Tyr
245 250 255
GAC AAA CCG TAC TAC ATG CTG AAT CTG TAC GAT CCG AAC AAA TAC GTT 816 Asp Lvs Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val ' 260 265 270
GAC GTC AAC AAT GTA GGT ATC CGC GGT TAC ATG TAC CTG AAA GGT CCG 864 Asp Val Asn Asn Val Gly He Arg Gly Tyr Met Tyr Leu Lys Gly Pro 275 280 ' 285
CGT GGT TCT GTT ATG ACT ACC AAC ATC TAC CTG AAC TCT TCC CTG TAC 912 Arg Gly Ser Val Met Thr Thr Asn He Tyr Leu Asn Ser Ser Leu Tyr 290 295 300 CGT GGT ACC AAA TTC ATC ATC AAG AAA TAC GCG TCT GGT AAC AAG GAC 960 Arq Glv Thr Lys Phe He He Lys Lys Tyr Ala Ser Gly Asn Lyε Asp 305 310 315 320
AAT ATC GTT CGC AAC AAT GAT CGT GTA TAC ATC AAT GTT GTA GTT AAG 1008 Asn He Val Arg Asn Asn Asp Arg Val Tyr He Asn Val Val Val Lys
325 330 335
AAC AAA GAA TAC CGT CTG GCT ACC AAT GCT TCT CAG GCT GGT GTA GAA 1056 Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala Gly Val Glu 340 345 350
AAG ATC TTG TCT GCT CTG GAA ATC CCG GAC GTT GGT AAT CTG TCT CAG 1104 Lys He Leu Ser Ala Leu Glu He Pro Asp Val Gly Asn Leu Ser Gin 355 360 365
GTA GTT GTA ATG AAA TCC AAG AAC GAC CAG GGT ATC ACT AAC AAA TGC 1152 Val Val Val Met Lys Ser Lys Asn Asp Gin Gly He Thr Asn Lys Cys 370 375 380 AAA ATG AAT CTG CAG GAC AAC AAT GGT AAC GAT ATC GGT TTC ATC GGT 1200 Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp He Gly Phe He Gly 385 390 395 400
TTC CAC CAG TTC AAC AAT ATC GCT AAA CTG GTT GCT TCC AAC TGG TAC 1248 Phe His Gin Phe Asn Asn He Ala Lys Leu Val Ala Ser Asn Trp Tyr 405 410 415
AAT CGT CAG ATC GAA CGT TCC TCT CGC ACT CTG GGT TGC TCT TGG GAG 1296 Asn Arg Gin He Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu 420 425 430
TTC ATC CCG GTT GAT GAC GGT TGG GGT GAA CGT CCG CTG TAACCCGGGA 1345 Phe He Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 435 440 445
ΛAGCTT 1351
(2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 445 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear (ri) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
Met His His His His His His Met Ala Arg Leu Leu Ser Thr Phe Thr 1 5 10 15
Glu Tyr He Lys Asn He He Asn Thr Ser He Leu Asn Leu Arg Tyr 20 25 30 Glu Ser Asn His Leu He Asp Leu Ser Arg Tyr Ala Ser Lys He Asn 35 40 45
He Gly Ser Lys Val Asn Phe Asp Pro He Asp Lys Asn Gin He Gin 50 55 60
Leu Phe Asn Leu Glu Ser Ser Lys He Glu Val He Leu Lys Asn Ala 65 70 75 80
He Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser Phe Trp He 85 90 95
Arg He Pro Lys Tyr Phe Asn Ser He Ser Leu Asn Asn Glu Tyr Thr 100 105 110 He He Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val Ser Leu Asn 115 120 125
Tyr Gly Glu He He Trp Thr Leu Gin Asp Thr Gin Glu He Lyε Gin 130 135 140
Arg Val Val Phe Lys Tyr Ser Gin Met He Asn He Ser Asp Tyr He 145 150 155 160
Asn Arg Trp He Phe Val Thr He Thr Asn Asn Arg Leu Asn Asn Ser 165 170 175
Lys He Tyr He Asn Gly Arg Leu He Asp Gin Lys Pro He Ser Asn 180 185 190 Leu Gly Asn He His Ala Ser Aεn Asn He Met Phe Lys Leu Asp Gly 195 200 205
Cys Arg Asp Thr His Arg Tyr He Trp He Lys Tyr Phe Asn Leu Phe
210 215 220
Asp Lys Glu Leu Asn Glu Lys Glu He Lys Asp Leu Tyr Asp Asn Gin
225 230 235 240
Ser Asn Ser Gly He Leu Lys Asp Phe Trp Gly Asp Tyr Leu Gin Tyr
245 250 255 Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val 260 265 ' 270
Asp Val Asn Asn Val Gly He Arg Gly Tyr Met Tyr Leu Lys Gly Pro 275 280 285
Arg Gly Ser Val Met Thr Thr Asn He Tyr Leu Asn Ser Ser Leu Tyr 290 295 300 Arg Gly Thr Lys Phe He He Lys Lys Tyr Ala Ser Gly Asn Lys Asp 305 310 315 320
Asn He Val Arg Asn Asn Asp Arg Val Tyr He Asn Val Val Val Lys 325 330 335
Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gin Ala Gly Val Glu 340 345 350
Lys He Leu Ser Ala Leu Glu He Pro Asp Val Glv Asn Leu Ser Gin 355 360 ' 365
Val Val Val Met Lys Ser Lys Asn Asp Gin Gly He Thr Asn Lys Cys 370 375 ' 380 Lys Met Asn Leu Gin Asp Asn Asn Gly Asn Asp He Gly Phe He Glv 385 390 395 400
Phe His Gin Phe Asn Asn He Ala Lys Leu Val Ala Ser Asn Trp Tyr 405 410 415
Aεn Arg Gin He Glu Arg Ser Ser Arg Thr Leu Gly Cys Ser Trp Glu 420 425 430
Phe He Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu 435 ' 440 445
(2) INFORMATION FOR SEQ ID NO : 37 :
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear in) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION, /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 37 : CGCATATGAA TATTCGTCCA TTGCATG 27
(2) INFORMATION FOR SEQ ID NO: 38:
( 1 ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear Ui) MOLECULE TYPE: other nucleic acid
• (A) DESCRIPTION: /desc = "DNA"
(κi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: GGAAGCTTGC AGGGCAATTA CATCATG 27
(2) INFORMATION FOR SEQ ID NO : 3 :
(1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3876 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3873
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 39 :
ATG CCA GTT ACA ATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48 Met Pro Val Thr He Asn Asn Phe Asn Tyr Asn Asp Pro He Asp Asn 1 5 10 15
GAC AAT ATT ATT ATG ATG GAA CCT CCA TTT GCA AGG GGT ACG GGG AGA 96 Asp Asn He He Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30
TAT TAT AAA GCT TTT AAA ATC ACA GAT CGT ATT TGG ATA ATA CCC GAA 144 Tyr Tyr Lys Ala Phe Lys He Thr Asp Arg He Trp He He Pro Glu 35 40 45 AGA TAT ACT TTT GGA TAT AAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60
ATT TTT AAT AGA GAT GTT TGT GAA TAT TAT GAT CCA GAT TAC TTA AAT 240 He Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80
ACC AAT GAT AAA AAG AAT ATA TTT TTC CAA ACA TTG ATC AAG TTA TTT 288 Thr Asn Asp Lys Lvs Asn He Phe Phe Gin Thr Leu He Lys Leu Phe 85 90 95
AAT AGA ATC AAA TCA AAA CCA TTG GGT GAA AAG TTA TTA GAG ATG ATT 336
Asn Arg He Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met He 100 * 105 ' 110
ATA AAT GGT ATA CCT TAT CTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384
He Asn Gly He Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 TTT AAC ACA AAC ATT GCT AGT GTA ACT GTT AAT AAA TTA ATT AGT AAT 432 Phe Asn Thr Asn He Ala Ser Val Thr Val Asn Lys Leu He Ser Asn 130 135 140
CCA GGA GAA GTG GAG CGA AAA AAA GGT ATT TTC GCA AAT TTA ATA ATA 480 Pro Gly Glu Val Glu Arg Lys Lys Gly He Phe Ala Asn Leu He He 145 150 155 160
TTT GGA CCT GGG CCA GTT TTA AAT GAA AAT GAG ACT ATA GAT ATA GGT 528 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr He Asp He Gly 165 170 175
ATA CAA AAT CAT TTT GCA TCA AGG GAA GGC TTT GGG GGT ATA ATG CAA 576
He Gin Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly He Met Gin 180 185 190
ATG AAA TTT TGT CCA GAA TAT GTA AGC GTA TTT AAT AAT GTT CAA GAA 624
Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gin Glu
195 ' 200 205 AAC AAA GGC GCA AGT ATA TTT AAT AGA CGT GGA TAT TTT TCA GAT CCA 672 Asn Lys Gly Ala Ser He Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220
GCC TTG ATA TTA ATG CAT GAA CTT ATA CAT GTT TTG CAT GGA TTA TAT 720 Ala Leu He Leu Met His Glu Leu He His Val Leu His Gly Leu Tyr 225 230 235 240
GGC ATT AAA GTA GAT GAT TTA CCA ATT GTA CCA AAT GAA AAA AAA TTT 768 Gly He Lys Val Asp Asp Leu Pro He Val Pro Asn Glu Lys Lys Phe 245 250 255
TTT ATG CAA TCT ACA GAT ACT ATA CAG GCA GAA GAA CTA TAT ACA TTT 816 Phe Met Gin Ser Thr Asp Thr He Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270
GGA GGA CAA GAT CCC AGC ATC ATA TCT CCT TCT ACA GAT AAA AGT ATC 864 Gly Gly Gin Asp Pro Ser He He Ser Pro Ser Thr Asp Lys S.er He 275 280 285 TAT GAT AAA GTT TTG CAA AAT TTT AGG GGG ATA GTT GAT AGA CTT AAC 912 Tyr Asp Lys Val Leu Gin Asn Phe Arg Gly He Val Asp Arg Leu Asn 290 295 300
AAG GTT TTA GTT TGC ATA TCA GAT CCT AAC ATT AAC ATT AAT ATA TAT 960 Lys Val Leu Val Cys He Ser Asp Pro Asn He Asn He Asn He Tyr 305 310 315 320
AAA AAT AAA TTT AAA GAT AAA TAT AAA TTC GTT GAA GAT TCT GAA GGA 1008 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly " 325 ' 330 335
AAA TAT AGT ATA GAT GTA GAA AGT TTC AAT AAA TTA TAT AAA AGC TTA 1056
Lys Tyr Ser He Asp Val Glu Ser Phe Aεn Lys Leu Tyr Lys Ser Leu
340 345 350
ATG TTA GGT TTT ACA GAA ATT AAT ATA GCA GAA AAT TAT AAA ATA AAA 1104
Met Leu Gly Phe Thr Glu He Asn He Ala Glu Asn Tyr Lys He Lys 355 360 365 ACT AGA GCT TCT TAT TTT AGT GAT TCC TTA CCA CCA GTA AAA ATA AAA 1152 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys He Lyε 370 375 380
AAT TTA TTA GAT AAT GAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200 Asn Leu Leu Asp Asn Glu He Tyr Thr He Glu Glu Gly Phe Asn He 385 390 395 400
TCT GAT AAA AAT ATG GGA AAA GAA TAT AGG GGT CAG AAT AAA GCT ATA 1248 Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gin Asn Lys Ala He 405 410 415
AAT AAA CAA GCT TAT GAA GAA ATC AGC AAG GAG CAT TTG GCT GTA TAT 1296
Asn Lys Gin Ala Tyr Glu Glu He Ser Lys Glu His Leu Ala Val Tyr
420 ' 425 430
AAG ATA CAA ATG TGT AAA AGT GTT AAA GTT CCA GGA ATA TGT ATT GAT 1344
Lys He Gin Met Cys Lys Ser Val Lys Val Pro Gly He Cys He Asp
435 440 445 GTC GAT AAT GAA AAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392 Val Asp Asn Glu Asn Leu Phe Phe He Ala Asp Lys Aεn Ser Phe Ser 450 455 460
GAT GAT TTA TCT AAA AAT GAA AGA GTA GAA TAT AAT ACA CAG AAT AAT 1440 Asp Asp Leu Ser Lys Asn Glu Arg Val Glu Tyr Asn Thr Gin Asn Asn 465 - 470 475 480
TAT ATA GGA AAT GAC TTT CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488 Tyr He Gly Asn Asp Phe Pro He Asn Glu Leu He Leu Asp Thr Asp 485 490 495
TTA ATA AGT AAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536
Leu He Ser Lys He Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
500 505 510 GAT TTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gin Pro Ala He Lys 515 520 " 525
AAA GTT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632 Lys Val Phe Thr Asp Glu Asn Thr He Phe Gin Tyr Leu Tyr Ser Gin 530 535 540
ACA TTT CCT CTA AAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680 Thr Phe Pro Leu Asn He Arg Asp He Ser Leu Thr Ser Ser Phe Asp 545 550 555 560
GAT GCA TTA TTA GTT TCT AGC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728 Asp Ala Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575
TAT ATT AAA ACT GCT AAT AAA GTA GTA GAA GCA GGA TTA TTT GCA GGT 1776 Tyr He Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590
TGG GTG AAA CAG ATA GTA GAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824 Trp Val Lys Gin He Val Aεp Asp Phe Val He Glu Ala Asn Lys Ser 595 600 605 AGT ACT ATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872 Ser Thr Met Asp Lys He Ala Asp He Ser Leu He Val Pro Tyr He 610 615 620
GGA TTA GCT TTA AAT GTA GGA GAT GAA ACA GCT AAA GGA AAT TTT GAA 1920 Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640
AGT GCT TTT GAG ATT GCA GGA TCC AGT ATT TTA CTA GAA TTT ATA CCA 1968 Ser Ala Phe Glu He Ala Gly Ser Ser He Leu Leu Glu Phe He Pro 645 650 655
GAA CTT TTA ATA CCT GTA GTT GGA GTC TTT TTA TTA GAA TCA TAT ATT 2016
Glu Leu Leu He Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr He
660 665 670
GAC AAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064
Asp Asn Lys Asn Lys He He Lys Thr He Asp Asn Ala Leu Thr Lys 675 680 685 AGA GTG GAA AAA TGG ATT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112 Arg Val Glu Lys Trp He Asp Met Tyr Gly Leu He Val Ala Gin Trp 690 695 700
CTC TCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160 Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr He Lys Glu Gly Met Tyr 705 710 715 720
AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC 2208 Lys Ala Leu Asn Tyr Gin Ala Gin Ala Leu Glu Glu He He Lys Tyr 725 730 735
AAA TAT AAT ATA TAT TCT GAA GAG GAA AAG TCA AAT ATT AAC ATC AAT 2256 Lys Tyr Asn He Tyr Ser Glu Glu Glu Lys Ser Asn He Asn He Asn 740 745 750
TTT AAT GAT ATA AAT TCT AAA CTT AAT GAT GGT ATT AAC CAA GCT ATG 2304 Phe Asn Asp He Asn Ser Lys Leu Asn Asp Gly He Asn Gin Ala Met 755 760 ' 765 GAT AAT ATA AAT GAT TTT ATA AAT GAA TGT TCT GTA TCA TAT TTA ATG 2352 Asp Asn He Asn Asp Phe He Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780
AAA AAA ATG ATT CCA TTA GCT GTA AAA AAA TTA CTA GAC TTT GAT AAT 2400 Lys Lys Met He Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785 790 795 800
ACT CTC AAA AAA AAT TTA TTA AAT TAT ATA GAT GAA AAT AAA TTA TAT 2448 Thr Leu Lys Lys Asn Leu Leu Asn Tyr He Asp Glu Asn Lys Leu Tyr 805 810 815
TTA ATT GGA AGT GTA GAA GAT GAA AAA TCA AAA GTA GAT AAA TAC TTG 2496
Leu He Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu
820 825 830
AAA ACC ATT ATA CCA TTT GAT CTT TCA ACG TAT TCT AAT ATT GAA ATA 2544
Lys Thr He He Pro Phe Asp Leu Ser Thr Tyr Ser Asn He Glu He
835 840 845 CTA ATA AAA ATA TTT AAT AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT 2592 Leu He Lys He Phe Asn Lys Tyr Asn Ser Glu He Leu Asn Asn He 850 855 860
ATC TTA AAT TTA AGA TAT AGA GAT AAT AAT TTA ATA GAT TTA TCA GGA 2640 He Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu He Asp Leu Ser Gly 865 870 875 880
TAT GGA GCA AAG GTA GAG GTA TAT GAT GGG GTC AAG CTT AAT GAT AAA 2688 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys 885 890 895
AAT CAA TTT AAA TTA ACT AGT TCA GCA GAT AGT AAG ATT AGA GTC ACT 2736
Asn Gin Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys He Arg Val Thr 900 905 910
CAA AAT CAG AAT ATT ATA TTT AAT AGT ATG TTC CTT GAT TTT AGC GTT 2784
Gin Asn Gin Asn He He Phe Asn Ser Met Phe Leu Asp Phe Ser Val 915 920 925 AGC TTT TGG ATA AGG ATA CCT AAA TAT AGG AAT GAT GAT ATA CAA AAT 2832 Ser Phe Trp He Arg He Pro Lys Tyr Arg Asn Asp Asp He Gin Asn 930 935 ' 940
TAT ATT CAT AAT GAA TAT ACG ATA ATT AAT TGT ATG AAA AAT AAT TCA 2880 Tyr He His Asn Glu Tyr Thr He He Asn Cys Met Lys Asn Asn Ser 945 950 955 960
GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGG ACC TTA ATT 2928 Gly Trp Lys He Ser He Arg Gly Asn Arg He He Trp Thr Leu He * 965 970 975
GAT ATA AAT GGA AAA ACC AAA TCA GTA TTT TTT GAA TAT AAC ATA AGA 2976
Asp He Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn He Arg 980 985 990
GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT 3024
Glu Asp He Ser Glu Tyr He Asn Arg Trp Phe Phe Val Thr He Thr 995 1000 1005 AAT AAT TTG GAT AAT GCT AAA ATT TAT ATT AAT GGC ACG TTA GAA TCA 3072 Asn Asn Leu Asp Asn Ala Lys He Tyr He Asn Gly Thr Leu Glu Ser 1010 1015 1020
AAT ATG GAT ATT AAA GAT ATA GGA GAA GTT ATT GTT AAT GGT GAA ATA 3120 Asn Met Asp He Lys Asp He Gly Glu Val He Val Asn Gly Glu He 1025 • 1030 1035 1040
ACA TTT AAA TTA GAT GGT GAT GTA GAT AGA ACA CAA TTT ATT TGG ATG 3168 Thr Phe Lys Leu Asp Gly Aεp Val Asp Arg Thr Gin Phe He Trp Met 1045 1050 1055
AAA TAT TTT AGT ATT TTT AAT ACG CAA TTA AAT CAA TCA AAT ATT AAA 3216
Lys Tyr Phe Ser He Phe Asn Thr Gin Leu Asn Gin Ser Asn He Lys 1060 1065 1070 GAG ATA TAT AAA ATT CAA TCA TAT AGC GAA TAC TTA AAA GAT TTT TGG 3264 Glu He Tyr Lys He Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 GGA AAT CCT TTA ATG TAT AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 3312 Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 ' 1095 ' 1100
AAT AAA AAT TCA TAT ATT AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA 3360 Asn Lys Asn Ser Tyr He Lys Leu Val Lys Asp Ser Ser Val Gly Glu 1105 ' 1110 1115 1120
ATA TTA ATA CGT AGC AAA TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT 3408 He Leu He Arg Ser Lys Tyr Asn Gin Asn Ser Asn Tyr He Asn Tyr 1125 1130 1135
AGA AAT TTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT 3456
Arg Asn Leu Tyr He Gly Glu Lys Phe He He Arg Arg Glu Ser Asn
1140 1145 1150
TCT CAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT 3504
Ser Gin Ser He Asn Asp Asp He Val Arg Lys Glu Asp Tyr He His
1155 1160 1165 CTA GAT TTG GTA CTT CAC CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA 3552 Leu Asp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys 1170 1175 1180
TAT TTT AAG GAA CAG GAA GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT 3600 Tyr Phe Lys Glu Gin Glu Glu Lys Leu Phe Leu Ser He He Ser Asp 1185 1190 1195 1200
TCT AAT GAA TTT TAT AAG ACT ATA GAA ATA AAA GAA TAT GAT GAA CAG 3648 Ser Asn Glu Phe Tyr Lys Thr He Glu He Lys Glu Tyr Asp Glu Gin 1205 1210 ' 1215
CCA TCA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT 3696
Pro Ser Tyr Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 * 1230
GAT GAT ATA GGA TTG ATT GGT ATT CAT CGT TTC TAC GAA TCT GGA GTT 3744
Asp Asp He Gly Leu He Gly He His Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245 TTA CGT AAA AAG TAT AAA GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA 3792 Leu Arg Lys Lys Tvr Lys Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu 1250 " ' 1255 1260
AAA GAG GTA AAA AGG AAA CCA TAT AAG TCA AAT TTG GGA TGT AAT TGG 3840 Lys Glu Val Lys Arg Lyε Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp 1265 1270 1275 1280
CAG TTT ATT CCT AAA GAT GAA GGG TGG ACT GAA TAA 3876
Gin Phe He Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
(2) INFORMATION FOR SEQ ID NO : 40 :
U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1291 ammo acids
Figure imgf000289_0001
(D) TOPOLOGY: linear
U i ) MOLECULE TYPE : protem
( Xl ) SEQUENCE DESCRI PTION : SEQ ID NO : 40 :
Met Pro Va l Thr H e Asn Asn Phe Asn Tyr Asn Asp Pro H e Asp Asn
1 5 10 15 Asp Asn He He Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30
Tyr Tyr Lys Ala Phe Lys He Thr Asp Arg He Trp He He Pro Glu 5 35 40 45
Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60
10 He Phe Asn Arg Asp Val Cys Glu Tyr Tyr Aεp Pro Asp Tyr Leu Asn 65 70 ' 75 80
Thr Asn Asp Lys Lys Asn He Phe Phe Gin Thr Leu He Lys Leu Phe 85 90 95
15
Asn Arg He Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met He 100 105 110
He Asn Gly He Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 0 115 120 125
Phe Asn Thr Asn He Ala Ser Val Thr Val Asn Lys Leu He Ser Asn 130 135 140
25 Pro Gly Glu Val Glu Arg Lyε Lys Gly He Phe Ala Asn Leu He He 145 150 155 160
Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr He Asp He Gly 165 170 175 )
:ie Gin Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly He Met Gin 180 185 190
Met Ly: Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gin Glu
195 200 205
Asn Lys Gly Ala Ser He Phe Asn Arg Arg Gly Tyr Phe Ser Aεp Pro 210 215 220
40 Ala Leu He Leu Met Hiε Glu Leu He Hiε Val Leu His Gly Leu Tyr 225 230 235 240
Gly He Lys Val Asp Asp Leu Pro He Val Pro Asn Glu Lys Lys Phe
245 250 255
45
Phe Met Gin Ser Thr Asp Thr He Gin Ala Glu Glu Leu Tyr Thr Phe
260 265 270
Gly Gly Gin Asp Pro Ser He He Ser Pro Ser Thr Asp Lys Ser He
50 275 280 285
Tyr Asp Lys Val Leu Gin Asn Phe Arg Gly He Val Asp Arg Leu Asn 290 295 300
Lys Val Leu Val Cys He Ser Asp Pro Aεn He Asn He Aεn He Tyr
305 310 315 320
Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335
60
Lys Tyr Ser He Asp Val Glu Ser Phe Asn Lyε Leu Tyr Lys Ser Leu 340 345 350
Met Leu Gly Phe Thr Glu He Asn He Ala Glu Asn Tyr Lys He Lys 65 355 360 365
Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys He Lys 370 375 380
70 Asn Leu Leu Asp Aεn Glu He Tyr Thr He Glu Glu Gly Phe Aεn He 385 390 395 400
Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gin Asn Lys Ala He 405 410 415
Asn Lys Gin Ala Tyr Glu Glu He Ser Lys Glu His Leu Ala Val Tyr 420 425 * 430
Lys He Gin Met Cys Lys Ser Val Lys Val Pro Gly He Cys He Asp 435 * 440 445
Val Asp Asn Glu Asn Leu Phe Phe He Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Val Glu Tyr Asn Thr Gin Asn Asn 465 470 475 480
Tyr He Gly Asn Asp Phe Pro He Asn Glu Leu He Leu Asp Thr Asp 485 490 495
Leu He Ser Lys He Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510
Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gin Pro Ala He Lys 515 520 525
Lys Val Phe Thr Asp Glu Asn Thr He Phe Gin Tyr Leu Tyr Ser Gin 530 535 540 Thr Phe Pro Leu Asn He Arg Asp He Ser Leu Thr Ser Ser Phe Asp
545 550 " 555 560
Asp Ala Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Aεp 565 570 575
Tyr He Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590
Trp Val Lys Gin He Val Asp Asp Phe Val He Glu Ala Asn Lyε Ser 595 600 605
Ser Thr Met Asp Lys He Ala Asp He Ser Leu He Val Pro Tyr He 610 615 620 Gly Leu Ala Leu Aεn Val Gly Aεp Glu Thr Ala Lys Gly Asn Phe Glu 625 630 ' ' 635 640
Ser Ala Phe Glu He Ala Gly Ser Ser He Leu Leu Glu Phe He Pro 645 650 655
Glu Leu Leu He Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr He 660 665 670
Asp Asn Lys Asn Lys He He Lys Thr He Asp Asn Ala Leu Thr Lys 675 680 685
Arg Val Glu Lys Trp He Asp Met Tyr Gly Leu He Val Ala Gin Trp 690 ' 695 " 700 Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr He Lys Glu Gly Met Tyr 705 710 715 720
Lys Ala Leu Asn Tyr Gin Ala Gin Ala Leu Glu Glu He He Lys Tyr 725 730 735
Lys Tyr Asn He Tyr Ser Glu Glu Glu Lys Ser Asn He Asn He Asn 740 745 750
Phe Asn Asp He Asn Ser Lys Leu Asn Asp Gly He Asn Gin Ala Met 755 760 765 Asp Asn He Asn Asp Phe He Asn Glu Cys Ser Val Ser Tyr Leu Met
770 775 780
Lys Lys Met He Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785 790 795 800
Thr Leu Lys Lys Asn Leu Leu Asn Tyr He Asp Glu Asn Lys Leu Tyr
805 810 815 Leu He Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu
820 825 830
Lys Thr He He Pro Phe Asp Leu Ser Thr Tyr Ser Asn He Glu He 835 840 845
Leu He Lys He Phe Asn Lys Tyr Asn Ser Glu He Leu Asn Asn He 850 855 860
He Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu He Asp Leu Ser Gly 865 870 875 880
Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys 885 890 895 Asn Gin Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys He Arg Val Thr
900 905 910
Gin Asn Gin Asn He He Phe Aεn Ser Met Phe Leu Asp Phe Ser Va l
915 920 925
Ser Phe Trp He Arg He Pro Lys Tyr Arg Asn Asp Asp He Gin Asn 930 " 935 940
Tyr He His Asn Glu Tyr Thr He He Asn Cys Met Lys Asn Asn Ser 945 950 955 960
Gly Trp Lys He Ser He Arg Gly Asn Arg He He Trp Thr Leu He 965 970 975 Asp lie Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn He Arg
980 985 990
Liu Asp He Ser Glu Tyr He Asn Arg Trp Phe Phe Val Thr He Thr 995 1000 1005
'.sn Asn Leu Asp Asn Ala Lys He Tyr He Asn Gly Thr Leu Glu Ser 1010 1015 ' 1020
Aεn Met Asp He Lys Aεp He Gly Glu Val He Val Aεn Gly Glu He 1025 1030 1035 1040
Thr Phe Lys Leu Asp Gly Asp Val Asp Arg Thr Gin Phe He Trp Met 1045 1050 1055 Lys Tyr Phe Ser He Phe Asn Thr Gin Leu Asn Gin Ser Asn He Lys
1060 1065 1070
Glu He Tyr Lys He Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085
Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly
1090 1095 1100
Asn Lys Asn Ser Tyr He Lys Leu Val Lyε Asp Ser Ser Val Gly Glu 1105 1110 1115 1120
He Leu He Arg Ser Lys Tyr Asn Gin Asn Ser Asn Tyr He Asn Tyr 1125 ' ' 1130 1135 Arg Asn Leu Tyr He Gly Glu Lys Phe He He Arg Arg Glu Ser Asn 1140 1145 1150
Ser Gin Ser He Asn Asp Asp He Val Arg Lys Glu Asp Tyr He His 1155 1160 ' 1165
Leu Asp Leu Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys 1170 1175 1180
Tyr Phe Lys Glu Gin Glu Glu Lys Leu Phe Leu Ser He He Ser Asp 1185 1190 1195 1200
Ser Asn Glu Phe Tyr Lys Thr He Glu He Lys Glu Tyr Asp Glu Gin 1205 1210 1215
Pro Ser Tyr Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230
Asp Asp He Gly Leu He Gly He His Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245
Leu Arg Lys Lys Tyr Lys Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu 1250 1255 1260
Lys Glu Val Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp 1265 1270 1275 ' 1280
Gin P.ne UP Pro Lvs Asp Glu Gly Trp Thr Glu 1285 1290 '2) INFORMATION FOR SEQ ID NO : 41.
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3876 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY linear
Ui) MOLECULE TYPE: DNA (genomic) (ιχ; FEATURE.
(A) NAME/KEY- CDS
(B) LOCATION. 1 .3873
(xi ) SEQUENCE DESCRIPTION SEQ ID NO .41
ATG CCA GTT ACA ATA AAT AAT TTT AAT TAT AAT GAT CCT ATT GAT AAT 48
Met Pro Val Thr He Asn Asn Phe Asn Tyr Asn Asp Pro He Asp Asn 1 5 10 15 AAT AAT ATT ATT ATG ATG GAG CCT CCA TTT GCG AGA GGT ACG GGG AGA 96
Aεn Asn He He Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30
TAT TAT AAA GCT TTT AAA ATC ACA GAT CGT ATT TGG ATA ATA CCG GAA 144 Tyr Tyr Lys Ala Phe Lys He Thr Asp Arg He Trp He He Pro Glu
35 40 45
AGA TAT ACT TTT GGA TAT AAA CCT GAG GAT TTT AAT AAA AGT TCC GGT 192
Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60
ATT TTT AAT AGA GAT GTT TGT GAA TAT TAT GAT CCA GAT TAC TTA AAT 240
He Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80
ACT AAT GAT AAA AAG AAT ATA TTT TTA CAA ACA ATG ATC AAG TTA TTT 288
Thr Asn Asp Lys Lys Asn He Phe Leu Gin Thr Met He Lys Leu Phe 85 90 95 AAT AGA ATC AAA TCA AAA CCA TTG GGT GAA AAG TTA TTA GAG ATG ATT 336 Asn Arg He Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met He 100 105 110
ATA AAT GGT ATA CCT TAT CTT GGA GAT AGA CGT GTT CCA CTC GAA GAG 384 He Asn Gly He Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 * 120 125
TTT AAC ACA AAC ATT GCT AGT GTA ACT GTT AAT AAA TTA ATC AGT AAT 432 Phe Asn Thr Asn He Ala Ser Val Thr Val Asn Lys Leu He Ser Asn 130 135 140
CCA GGA GAA GTG GAG CGA AAA AAA GGT ATT TTC GCA AAT TTA ATA ATA 480 Pro Gly Glu Val Glu Arg Lys Lys Gly He Phe Ala Asn Leu He He 145 150 155 160
TTT GGA CCT GGG CCA GTT TTA AAT GAA AAT GAG ACT ATA GAT ATA GGT 528 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr He Asp He Gly 165 170 175 ATA CAA AAT CAT TTT GCA TCA AGG GAA GGC TTC GGG GGT ATA ATG CAA 576 He Gin Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly He Met Gin 180 185 190
ATG AAG TTT TGC CCA GAA TAT GTA AGC GTA TTT AAT AAT GTT CAA GAA 624 Met Lyε Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gin Glu 195 200 205
.AAC AAA GGC GCA AGT ATA TTT AAT AGA CGT GGA TAT TTT TCA GAT CCA 672 Asn Lys Gly Ala Ser He Phe Asn Arg Arg Gly Tyr Phe Ser Aεp Pro 210 215 220
GCC TTG ATA TTA ATG CAT GAA CTT ATA CAT GTT TTA CAT GGA TTA TAT 720
Ala Leu He Leu Met His Glu Leu He Hiε Val Leu His Gly Leu Tyr 225 230 235 240
GGC ATT AAA GTA GAT GAT TTA CCA ATT GTA CCA AAT GAA AAA AAA TTT 768
Gly He Lys Val Asp Asp Leu Pro He Val Pro Asn Glu Lys Lys Phe 24 250 255 TTT ATG CAA TCT ACA GAT GCT ATA CAG GCA GAA GAA CTA TAT ACA TTT 816 Phe Met Gin Ser Thr Asp Ala He Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270
GGA GGA CAA GAT CCC AGC ATC ATA ACT CCT TCT ACG GAT AAA AGT ATC 864 Gly Gly Gin Asp Pro Ser He He Thr Pro Ser Thr Asp Lys Ser He 275 280 285
TAT GAT AAA GTT TTG CAA AAT TTT AGA GGG ATA GTT GAT AGA CTT AAC 912 Tyr Asp Lyε Val Leu Gin Asn Phe Arg Gly He Val Aεp Arg Leu Asn 290 ' 295 300
AAG GTT TTA GTT TGC ATA TCA GAT CCT AAC ATT AAT ATT AAT ATA TAT 960
Lys Val Leu Val Cys He Ser Asp Pro Asn He Asn He Asn He Tyr 305 310 315 320
AAA AAT AAA TTT AAA GAT AAA TAT AAA TTC GTT GAA GAT TCT GAG GGA 1008
Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 AAA TAT AGT ATA GAT GTA GAA AGT TTT GAT AAA TTA TAT AAA AGC TTA 1056 Lys Tyr Ser He Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350
ATG TTT GGT TTT ACA GAA ACT AAT ATA GCA GAA AAT TAT AAA ATA AAA 1104 Met Phe Gly Phe Thr Glu Thr Asn He Ala Glu Asn Tyr Lys He Lys 355 360 365
ACT AGA GCT TCT TAT TTT AGT GAT TCC TTA CCA CCA GTA AAA ATA AAA 1152 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys He Lys 370 375 380 AAT TTA TTA GAT AAT GAA ATC TAT ACT ATA GAG GAA GGG TTT AAT ATA 1200 Asn Leu Leu Asp Asn Glu He Tyr Thr He Glu Glu Gly Phe Asn He 385 390 395 400 TCT GAT AAA GAT ATG GAA AAA GAA TAT AGA GGT CAG AAT AAA GCT ATA 1248 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gin Asn Lys Ala He 405 410 " 415
AAT AAA CAA GCT TAT GAA GAA ATT AGC AAG GAG CAT TTG GCT GTA TAT 1296 Asn Lys Gin Ala Tyr Glu Glu He Ser Lys Glu His Leu Ala Val Tyr
420 425 430
AAG ATA CAA ATG TGT AAA AGT GTT AAA GCT CCA GGA ATA TGT ATT GAT 1344 Lys He Gin Met Cys Lys Ser Val Lys Ala Pro Gly He Cys He Asp 435 440 445
GTT GAT AAT GAA GAT TTG TTC TTT ATA GCT GAT AAA AAT AGT TTT TCA 1392
Val Asp Asn Glu Asp Leu Phe Phe He Ala Asp Lys Asn Ser Phe Ser 450 455 460
GAT GAT TTA TCT AAA AAC GAA AGA ATA GAA TAT AAT ACA CAG AGT AAT 1440
Asp Asp Leu Ser Lys Asn Glu Arg He Glu Tyr Asn Thr Gin Ser Asn 465 470 475 480 TAT ATA GAA AAT GAC TTC CCT ATA AAT GAA TTA ATT TTA GAT ACT GAT 1488 Tyr He Glu Asn Asp Phe Pro He Asn Glu Leu He Leu Asp Thr Asp 485 490 495
TTA ATA AGT AAA ATA GAA TTA CCA AGT GAA AAT ACA GAA TCA CTT ACT 1536 Leu He Ser Lys He Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
500 505 510
GAT TTT AAT GTA GAT GTT CCA GTA TAT GAA AAA CAA CCC GCT ATA AAA 1584 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gin Pro Ala He Lys 515 520 ' 525
AAA ATT TTT ACA GAT GAA AAT ACC ATC TTT CAA TAT TTA TAC TCT CAG 1632
Lys He Phe Thr Asp Glu Asn Thr He Phe Gin Tyr Leu Tyr Ser Gin
530 535 540
ACA TTT CTC TTA GAT ATA AGA GAT ATA AGT TTA ACA TCT TCA TTT GAT 1680
Thr Phe Leu Leu Asp He Arg Asp He Ser Leu Thr Ser Ser Phe Asp
545 550 555 560 GAT GCA TTA TTA TTT TCT AAC AAA GTT TAT TCA TTT TTT TCT ATG GAT 1728 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575
TAT ATT AAA ACT GCT AT AAA GTG GTA GAA GCA GGA TTA TTT GCA GGT 1776 Tyr He Lys Thr Ala Aεn Lyε Val Val Glu Ala Gly Leu Phe Ala Gly
580 585 590
TGG GTG AAA CAG ATA GTA AAT GAT TTT GTA ATC GAA GCT AAT AAA AGC 1824 Trp Val Lys Gin He Val Asn Asp Phe Val He Glu Ala Asn Lys Ser ' 595 600 605
AAT ACT ATG GAT AAA ATT GCA GAT ATA TCT CTA ATT GTT CCT TAT ATA 1872
Asn Thr Met Asp Lys He Ala Asp He Ser Leu He Val Pro Tyr He
610 615 620
GGA TTA GCT TTA AAT GTA GGA AAT GAA ACA GCT AAA GGA AAT TTT GAA 1920
Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu
625 630 635 640 AAT GCT TTT GAG ATT GCA GGA GCC AGT ATT CTA CTA GAA TTT ATA CCA 1968 Asn Ala Phe Glu He Ala Gly Ala Ser He Leu Leu Glu Phe He Pro 645 650 655
GAA CTT TTA ATA CCT GTA GTT GGA GCC TTT TTA TTA GAA TCA TAT ATT 2016 Glu Leu Leu He Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr He 660 665 6 70
GAC AAT AAA AAT AAA ATT ATT AAA ACA ATA GAT AAT GCT TTA ACT AAA 2064 Asp Asn Lys Asn Lys He He Lys Thr He Asp Asn Ala Leu Thr Lys 675 680 685
AGA AAT GAA AAA TGG AGT GAT ATG TAC GGA TTA ATA GTA GCG CAA TGG 2112 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu He Val Ala Gin Trp 690 695 700
CTC TCA ACA GTT AAT ACT CAA TTT TAT ACA ATA AAA GAG GGA ATG TAT 2160 Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr He Lys Glu Gly Met Tyr ^0 710 715 ' 720 AAG GCT TTA AAT TAT CAA GCA CAA GCA TTG GAA GAA ATA ATA AAA TAC 2208 Lys Ala Leu Asn Tyr Gin Ala Gin Ala Leu Glu Glu He He Lyε Tyr 725 730 735
AGA TAT AAT ATA TAT TCT GAA AAA GAA AAG TCA AAT ATT AAC ATC GAT 2256 Arg Tyr Asn He Tyr Ser Glu Lys Glu Lys Ser Asn He Asn He Asp
740 ' 745 750
TTT AAT GAT ATA AAT TCT AAA CTT AAT GAG GGT ATT AAC CAA GCT ATA 2304 Phe Asn Asp He Asn Ser Lys Leu Asn Glu Gly He Asn Gin Ala He 755 760 765
GAT AAT ATA AAT AAT TTT ATA AAT GGA TGT TCT GTA TCA TAT TTA ATG 2352 Asp Asn He Asn Asn Phe He Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780
AAA AAA ATG ATT CCA TTA GCT GTA GAA AAA TTA CTA GAC TTT GAT AAT 2400 Lys Lys Met He Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 ' 790 795 800 ACT CTC AAA AAA AAT TTG TTA AAT TAT ATA GAT GAA AAT AAA TTA TAT 2448 T.ir Leu Lys Lys Asn Leu Leu Asn Tyr He Asp Glu Asn Lys Leu Tyr 805 810 815
TTG ATT GGA AGT GCA GAA TAT GAA AAA TCA AAA GTA AAT AAA TAC TTG 2496 Leu He Gly Ser Ala Glu Tyr Glu Lyε Ser Lyε Val Asn Lys Tyr Leu
820 * 825 830
AAA ACC ATT ATG CCG TTT GAT CTT TCA ATA TAT ACC AAT GAT ACA ATA 2544 Lys Thr He Met Pro Phe Asp Leu Ser He Tyr Thr Asn Asp Thr He " 835 * 840 845
CTA ATA GAA ATG TTT AAT AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT 2592
Leu He Glu Met Phe Asn Lys Tyr Asn Ser Glu He Leu Asn Asn He 850 855 860
ATC TTA AAT TTA AGA TAT AAG GAT AAT AAT TTA ATA GAT TTA TCA GGA 2640
He Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu He Asp Leu Ser Gly 865 B70 875 880 TAT GGG GCA AAG GTA GAG GTA TAT GAT GGA GTC GAG CTT AAT GAT AAA 2688 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895
AAT CAA TTT AAA TTA ACT AGT TCA GCA AAT AGT AAG ATT AGA GTG ACT 2736 Asn Gin Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys He Arg Val Thr
900 905 910
CAA AAT CAG AAT ATC ATA TTT AAT AGT GTG TTC CTT GAT TTT AGC GTT 2784 Gin Asn Gin Asn He He Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925
AGC TTT TGG ATA AGA ATA CCT AAA TAT AAG AAT GAT GGT ATA CAA AAT 2832
Ser Phe Trp He Arg He Pro Lys Tyr Lys Asn Asp Gly He Gin Asn
930 935 940 TAT ATT CAT AAT GAA TAT ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG 2880
Tyr He His Asn Glu Tyr Thr He He Asn Cys Met Lys Asn Asn Ser 945 950 955 960
GGC TGG AAA ATA TCT ATT AGG GGT AAT AGG ATA ATA TGG ACT TTA ATT 2928
Gly Trp Lys He Ser He Arg Gly Asn Arg He He Trp Thr Leu He
965 ' 970 975
GAT ATA AAT GGA AAA ACC AAA TCG GTA TTT TTT GAA TAT AAC ATA AGA 2976
Asp He Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn He Arg 980 985 990
GAA GAT ATA TCA GAG TAT ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT 3024
Glu Asp He Ser Glu Tyr He Asn Arg Trp Phe Phe Val Thr He Thr 995 1000 1005
AAT AAT TTG AAT AAC GCT AAA ATT TAT ATT AAT GGT AAG CTA GAA TCA 3072
Asn Asn Leu Asn Asn Ala Lys He Tyr He Asn Gly Lys Leu Glu Ser 1010 1015 1020
AAT ACA GAT ATT AAA GAT ATA AGA GAA GTT ATT GCT AAT GGT GAA ATA 3120
Asn Thr Asp He Lys Asp He Arg Glu Val He Ala Aεn Gly Glu He 1025 1030 1035 1040 ATA TIT AAA TTA GAT GGT GAT ATA GAT AGA ACA CAA TTT ATT TGG ATG 3168
He Phe Lys Leu Asp Gly Asp He Asp Arg Thr Gin Phe He Trp Met
1045 1050 1055
AAA TAT TTC AGT ATT TTT AAT ACG GAA TTA AGT CAA TCA AAT ATT GAA 3216 Lys Tyr Phe Ser He Phe Asn Thr Glu Leu Ser Gin Ser Asn He Glu
1060 1065 1070
GAA AGA TAT AAA ATT CAA TCA TAT AGC GAA TAT TTA AAA GAT TTT TGG 3264
Glu Arg Tyr Lys He Gin Ser Tyr Ser Glu Tyr Leu Lys Aεp Phe Trp 1075 1080 1085
GGA AAT CCT TTA ATG TAC AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG 3312
Gly Aεn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 * 1095 1100
AAT AAA AAT TCA TAT ATT AAA CTA AAG AAA GAT TCA CCT GTA GGT GAA 3360
Asn Lys Asn Ser Tyr He Lys Leu Lys Lys Asp Ser Pro Val Gly Glu 1105 1110 " 1115 1120 ATT TTA ACA CGT AGC AAA TAT AAT CAA AAT TCT AAA TAT ATA AAT TAT 3408
He Leu Thr Arg Ser Lys Tyr Asn Gin Asn Ser Lys Tyr He Asn Tyr
1125 1130 ' 1135
AGA GAT TTA TAT ATT GGA GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT 3456 Arg Asp Leu Tyr He Gly Glu Lys Phe He He Arg Arg Lys Ser Asn
1140 1145 " 1150
TCT CAA TCT ATA AAT GAT GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT 3504
Ser Gin Ser He Asn Asp Asp He Val Arg Lys Glu Asp Tyr He Tyr 1155 1160 1165
CTA GAT TTT TTT AAT TTA AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA 3552
Leu Asp Phe Phe Asn Leu Asn Gin Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180
TAT TTT AAG AAA GAG GAA GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT 3600
Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro He Ser Asp 1185 ' 1190 1195 120C TCT GAT GAG TTT TAC AAT ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG 3648
Ser Asp Glu Phe Tyr Asn Thr He Gin He Lys Glu Tyr Asp Glu Gin
1205 1210 1215 CCA ACA TAT AGT TGT CAG TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT 3696 Pro Thr Tyr Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 * 1230 GAT GAG ATA GGA TTG ATT GGT ATT CAT CGT TTC TAC GAA TCT GGA ATT 3744 Asp Glu He Gly Leu He Gly He His Arg Phe Tyr Glu Ser Gly He 1235 1240 1245
GTA TTT GAA GAG TAT AAA GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA 3792 Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu 1250 1255 1260
AAA GAG GTA AAA AGG AAA CCA TAT AAT TTA AAA TTG GGA TGT AAT TGG 3640 Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp 1265 1270 1275 1280
CAG TTT ATT CCT AAA GAT GAA GGG TGG ACT GAA TAA 3876
Gin Phe He Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
(2) INFORMATION FOR SEQ ID NO : 2 :
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1291 ammo acids
Figure imgf000298_0001
(D) TOPOLOGY: linear in) MOLECULE TYPE: protem (κi) SEQUENCE DESCRIPTION: SEQ ID NO: 42.
Met Pro Val Thr He Asn Aεn Phe Asn Tyr Asn Asp Pro He Asp Asn 1 5 10 15 Asn Asn He He Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg
20 25 30
Tyr Tyr Lys Ala Phe Lys He Thr Asp Arg He Trp He He Pro Glu 35 40 45
Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Aεp Phe Aεn Lyε Ser Ser Gly 50 55 60
He Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Aεp Tyr Leu Λεn 65 ' 70 75 80
Thr Aεn Asp Lys Lys Asn He Phe Leu Gin Thr Met He Lys Leu Phe 85 90 95 Asn Arg He Lvs Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met He
100 105 110
He Asn Gly He Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu
115 120 125
Phe Asn Thr Asn He Ala Ser Val Thr Val Asn Lys Leu He Ser Asn
130 135 140
Pro Gly Glu Val Glu Arg Lys Lys Gly He Phe Ala Aεn Leu He He 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr He Asp He Gly 165 170 175
He Gin Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly He Met Gin 180 185 190
Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gin Glu 195 200 205 Asn Lys Gly Ala Ser He Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro
210 215 220
Ala Leu He Leu Met His Glu Leu He His Val Leu His Gly Leu Tyr 225 230 235 240
Gly He Lys Val Asp Asp Leu Pro He Val Pro Asn Glu Lys Lys Phe 245 250 255
Phe Met Gin Ser Thr Asp Ala He Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270
Gly Gly Gin Asp Pro Ser He He Thr Pro Ser Thr Asp Lys Ser He 275 280 285 "yr Asp Lys Val Leu Gin Asn Phe Arg Gly He Val Asp Arg Leu Asn 290 295 * 300
Lys Val Leu Val Cys He Ser Asp Pro Asn He Asn He Asn He Tyr
305 ' 310 315 320
Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335
Lys Tyr Ser He Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 ' 350
Met Phe Gly Phe Thr Glu Thr Asn He Ala Glu Asn Tyr Lys He Lys 355 360 365
Tnr Arg Ala Ser Tvr Phe Ser Asp Ser Leu Pro Pro Val Lys He Lys 370 375 380
Asn Leu Leu Asp Asn Glu He Tyr Thr He Glu Glu Gly Phe Asn He
385 390 * 395 400
Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Glv Gin Asn Lys Ala He
" 405 ' ' 410 " ' 41b
Asn Lys Gin Ala Tyr Glu Glu He Ser Lys Glu His Leu Ala Val Tyr 420 425 430
Lys He Gin Met Cys Lys Ser Val Lys Ala Pro Gly He Cys He Asp 435 ' 440 ' 445 Val Asp Asn Glu Asp Leu Phe Phe He Ala Asp Lyε Asn Ser Phe Ser 450 455 460
Asp Aεp Leu Ser Lys Asn Glu Arg He Glu Tyr Asn Thr Gin Ser Asn
465 470 475 480
Tyr He Glu Asn Asp Phe Pro He Asn Glu Leu He Leu Asp Thr Asp
485 490 495
Leu He Ser Lys He Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510
Aεp Phe Asn Val Aεp Val Pro Val Tyr Glu Lys Gin Pro Ala He Lvs
515 520 525 Lys He Phe Thr Asp Glu Asn Thr He Phe Gin Tyr Leu Tyr Ser Gin 530 535 540
Thr Phe Leu Leu Asp He Arg Asp He Ser Leu Thr Ser Ser Phe Asp 545 550 555 560
Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575
Tyr He Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590
Trp Val Lys Gin He Val Asn Asp Phe Val He Glu Ala Asn L.ys Ser 595 600 605
Asn Thr Met Asp Lys He Ala Asp He Ser Leu He Val Pro Tyr He 610 615 620
Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640
Asn Ala Phe Glu He Ala Gly Ala Ser He Leu Leu Glu Phe He Pro 645 650 655
Glu Leu Leu He Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr He 660 665 670
Asp Aεn Lys Asn Lys He He Lys Thr He Asp Asn Ala Leu Thr Lys 675 680 685 Ai g Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu He Val Ala Gin Trp 690 695 ' 700
Leu Sej Thr Val Asn Thr Gin Phe Tyr Thr He Lys Glu Giy Met Tyr
705 710 715 ' 720
Lys Ala Leu Asn Tyr Gin Ala Gin Ala Leu Glu Glu He He Lyε Tyr 725 730 735 '
Arg Tyr Asn He Tyr Ser Glu Lys Glu Lys Ser Asn He Asn He Aεp ' -740 745 750
Phe Asn Asp He Asn Ser Lys Leu Aεn Glu Gly He Λεn Gin Ala He 755 ' 760 ' 765 Asp Asn He Asn Aεn Phe He Asn Gly Cys Ser Val Ser Tyr Leu Met
770 775 780
Lys Lys Met He Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800
Thr Leu Lyε Lys Asn Leu Leu Aεn Tyr He Asp Glu Asn Lys Leu Tyr 805 810 815
Leu He Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830
Lys Thr He Met Pro Phe Asp Leu Ser He Tyr Thr Asn Asp Thr He 835 840 845 Leu He Glu Met Phe Asn Lys Tyr Asn Ser Glu He Leu Asn Aεn He 850 855 860
He Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu He Asp Leu Ser Gly
865 870 " 875 880
Tyr Gly Ala Lys Val Glu Val Tyr Aεp Gly Val Glu Leu Asn Asp Lys
885 890 895
Asn Gin Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys He Arg Val Thr 900 905 910 Gin Asn Gin Asn He He Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925
Ser Phe Trp He Arg He Pro Lys Tyr Lys Asn Asp Gly He Gin Asn 930 935 940
Tyr He His Asn Glu Tyr Thr He He Asn Cys Met Lys Asn Asn Ser 945 950 955 960
Gly Trp Lys He Ser He Arg Gly Asn Arg He He Trp Thr Leu He 965 970 975
Asp He Aεn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn He Arg 980 ' 985 990
Glu Asp He Ser Glu Tyr He Asn Arg Trp Phe Phe Val Thr He Thr 995 1000 1005
Asn Asn Leu Asn Asn Ala Lys He Tyr He Asn Gly Lys Leu Glu Ser 101O 1015 1020
Asn Thr Asp He Lys Asp He Arg Glu Val He Ala Asn Gly Glu He 1025 1030 1035 1040 He Pne Lys Leu Asp Gly Asp He Asp Arg Thr Gin Phe He Trp Met
1045 1050 1055
Lys Ty r Phe Ser He Phe Asn Thr Glu Leu Ser Gin Ser Asn He Glu 1060 1065 1070
Glu Arg Tyr Lys He Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 ' 1080 1085
Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090 1095 1100
Asn Lvs Asn Ser Tyr He Lys Leu Lys Lys Asp Ser Pro Val Gly Glu 1105 1110 ' 1115 1120 He Leu Thr Arg Ser Lys Tyr Asn Gin Asn Ser Lys Tyr He Asn Tyr
1125 1130 1135
Arg Asp Leu Tyr He Gly Glu Lys Phe He He Arg Arg Lys Ser Asn 1140 ' 1145 " 1150
;r Gin Ser He Asn Asp Asp He Val Arg Lys Glu Asp Tyr He Tvr 1155 1160 1165
Leu Asp Phe Phe Asn Leu Asn Gin Glu Trp Arg Val Tyr Thr Tyr Lys 1170 1175 1180
Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro He Ser Asp 1185 1190 1195 1200 Ser Asp Glu Phe Tyr Asn Thr He Gin He Lys Glu Tyr Asp Glu Gin
1205 1210 1215
Pro Thr Tyr Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230
Aεp Glu He Gly Leu He Gly He His Arg Phe Tyr Glu Ser Gly He 1235 1240 1245
Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu 1250 ' ' 1255 ' ' 1260
Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp 1265 1270 1275 1280 Gin Phe He Pro Lys Asp Glu Gly Trp Thr Glu 1285 1290
(2) INFORMATION FOR SEQ ID NO : 43 :
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1526 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS. double
(D) TOPOLOGY: linear
U l ) MOLECULE TYPE : other nuclei c acid
(A) DESCRIPTION: /desc = "DNA" l ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 108..1523
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 43 : AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Met Gly His 1
CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His Hrs Hiε His His His His His His Ser Ser Gly His He Glu Gly
5 10 15 CGT CAT ATG GCT AGC ATG GCT GAT ACA ATA CTA ATA GAA ATG TTT AAT 212 Arg Hiε Met Ala Ser Met Ala Asp Thr He Leu He Glu Met Phe Asn 20 25 30 35
AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT ATC TTA AAT TTA AGA TAT 260 Lys Tyr Asn Ser Glu He Leu Asn Asn He He Leu Asn Leu Arg Tyr
40 45 50
AGA GAT AAT AAT TTA ATA GAT TTA TCA GGA TAT GGA GCA AAG GTA GAG 308 Arg Asp Asn Asn Leu He Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu 55 60 65
GTA TAT GAT GGG GTC AAG CTT AAT GAT AAA AAT CAA TTT AAA TTA ACT 356 Val Tyr Asp Gly Val Lys Leu Asn Asp Lys Asn Gin Phe Lys Leu Thr 70 75 80
AGT TCA GCA GAT AGT AAG ATT AGA GTC ACT CAA AAT CAG AAT ATT ATA 404 Ser Ser Ala Asp Ser Lys He Arg Val Thr Gin Asn Gin Asn He He 85 90 95 TTT AAT AGT ATG TTC CTT GAT TTT AGC GTT AGC TTT TGG ATA AGG ATA 452 Phe Asn Ser Met Phe Leu Asp Phe Ser Val Ser Phe Trp He Arg He 100 105 110 ' 115
CCT AAA TAT AGG AAT GAT GAT ATA CAA AAT TAT ATT CAT AAT GAA TAT 500 Pro Lys Tvr Arg Asn Asp Asp He Gin Asn Tyr He His Asn Glu Tyr
120 125 130
ACG ATA ATT AAT TGT ATG AAA AAT AAT TCA GGC TGG AAA ATA TCT ATT 548 Thr He He Asn Cys Met Lys Asn Asn Ser Gly Trp Lys He Ser He 135 140 145
AGG GGT AAT AGG ATA ATA TGG ACC TTA ATT GAT ATA AAT GGA AAA ACC 596 Arg Gly Asn Arg He He Trp Thr Leu He Asp He Asn Gly Lys Thr 150 155 160
AAA TCA GTA TTT TTT GAA TAT AAC ATA AGA GAA GAT ATA TCA GAG TAT 644 Lys Ser Val Phe Phe Glu Tyr Asn He Arg Glu Asp He Ser Glu Tyr 165 170 175 ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT AAT AAT TTG GAT AAT GCT 692 He Asn Arg Trp Phe Phe Val Thr He Thr Asn Asn Leu Asp Asn Ala 180 185 190 195
AAA ATT TAT ATT AAT GGC ACG TTA GAA TCA AAT ATG GAT ATT AAA GAT 740
Lys He Tyr He Asn Gly Thr Leu Glu Ser Asn Met Asp He Lys Asp 200 205 210
ATA GGA GAA GTT ATT GTT AAT GGT GAA ATA ACA TTT AAA TTA GAT GGT 788
He Gly Glu Val He Val Asn Gly Glu He Thr Phe Lys Leu Asp Gly
215 220 225
GAT GTA GAT AGA ACA CAA TTT ATT TGG ATG AAA TAT TTT AGT ATT TTT 836
Asp Val Asp Arg Thr Gin Phe He Trp Met Lys lyr Phe Ser He Phe 230 235 ' ' 240
AAT ACG CAA TTA AAT CAA TCA AAT ATT AAA GAG ATA TAT AAA ATT CAA 884
Asn Thr Gin Leu Asn Gin Ser Asn He Lyε Glu He Tyr Lyε He Gin 245 250 255 TCA TAT AGC GAA TAC TTA AAA GAT TTT TGG GGA AAT CCT TTA ATG TAT 932
Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr 260 ' 265 270 275
AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG AAT AAA AAT TCA TAT ATT 980 Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lvs Asn Ser Tyr He
280 285 290
AAA CTA GTG AAA GAT TCA TCT GTA GGT GAA ATA TTA ATA CGT AGC AAA 1028
Lys Leu Val Lys Asp Ser Ser Val Gly Glu He Leu He Arg Ser Lvs ' 295 300 305
TAT AAT CAG AAT TCC AAT TAT ATA AAT TAT AGA AAT TTA TAT ATT GGA 1076
Tyr Asn Gin Asn Ser Aεn Tyr He Asn Tyr Arg Asn Leu Tyr He Gly 310 315 320
GAA AAA TTT ATT ATA AGA AGA GAG TCA AAT TCT CAA TCT ATA AAT GAT 1124
Glu Lys Phe He He Arg Arg Glu Ser Asn Ser Gin Ser He Aεn Asp 325 330 335 GAT ATA GTT AGA AAA GAA GAT TAT ATA CAT CTA GAT TTG GTA CTT CAC 1172
Asp He Val Arg Lys Glu Asp Tyr He His Leu Asp Leu Val Leu His 340 345 * 350 355
CAT GAA GAG TGG AGA GTA TAT GCC TAT AAA TAT TTT AAG GAA CAG GAA 1220 His Glu Glu Trp Arg Val Tyr Ala Tyr Lyε Tyr Phe Lys Glu Gin Glu
360 365 ' ' 370
GAA AAA TTG TTT TTA TCT ATT ATA AGT GAT TCT AAT GAA TTT TAT AAG 1268
Glu Lys Leu Phe Leu Ser He He Ser Asp Ser Asn Glu Phe Tyr Lys 375 3B0 385
ACT ATA GAA ATA AAA GAA TAT GAT GAA CAG CCA TCA TAT AGT TGT CAG 1316
Thr He Glu He Lys Glu Tyr Asp Glu Gin Pro Ser Tyr Ser Cys Gin 390 395 400
TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT GAT GAT ATA GGA TTG ATT 1364
Leu Leu Phe Lyε Lys Asp Glu Glu Ser Thr Asp Asp He Gly Leu He 405 410 415 GGT ATT CAT CGT TTC TAC GAA TCT GGA GTT TTA CGT AAA AAG TAT AAA 1412
Gly He His Arg Phe Tyr Glu Ser Gly Val Leu Arg Lys Lys Tyr Lys 420 425 ' 430 435
GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA AAA GAG GTA AAA AGG AAA 1460 Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys
440 445 450
CCA TAT AAG TCA AAT TTG GGA TGT AAT TGG CAG TTT ATT CCT AAA GAT 1508
Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp Gin Phe He Pro Lys Asp 455 460 465 GAA GGG TGG ACT GAA TAA 1526
Glu Gly Trp Thr Glu 470 (2) INFORMATION FOR SEQ ID NO: 44:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 472 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY- linear
Ui) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15
He Glu Gly Arg His Met Ala Ser Met Ala Asp Thr He Leu He Glu 20 25 30
Met Phe Asn Lys Tyr Asn Ser Glu He Leu Asn Asn He He Leu Asn 35 40 45 Leu Arg Tyr Arg Asp Asn Asn Leu He Aεp Leu Ser Gly Tyr Gly Ala 50 55 60
Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys Asn Gin Phe 65 70 75 80
Lys Leu Thr Ser Ser Ala Asp Ser Lys He Arg Val Thr Gin Asn Gin 85 90 95
Asn He He Phe Asn Ser Met Phe Leu Asp Phe Ser Val Ser Phe Trp 100 105 110
He Arg He Pro Lyε Tyr Arg Asn Asp Asp He Gin Asn Tyr He His
115 120 125 Asn Glu Tyr Thr He He Aεn Cys Met Lys Asn Asn Ser Gly Trp Lys 130 135 140
He Ser He Arg Gly Asn Arg He He Trp Thr Leu He Asp He Asn 145 150 155 L60
Thr Lys Ser Val Phe Phe Glu Tyr Asn LC Arg Glu Asp lit 165 170 175
Ser Glu Tyr He Asn Arg Trp Phe Phe Val Thr He Thr Asn Asn Leu 180 185 190
Asp Asn Ala Lys He Tyr He Asn Gly Thr Leu Glu Ser Asn Met Asp 195 200 205 He Lys Asp He Gly Glu Val He Val Asn Gly Glu He Thr Phe Lys 210 215 220
Leu Asp Gly Asp Val Asp Arg Thr Gin Phe He Trp Met Lys Tyr Phe
225 230 235 240
Ser He Phe Aεn Thr Gin Leu Asn Gin Ser Aεn He Lyε Glu He Tyr
245 250 255
Lyε He Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Aεn Pro ' 260 265 270
Leu Met Tyr Aεn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn 275 ' ' 280 285 Ser Tyr He Lys Leu Val Lyε Asp Ser Ser Val Gly Glu He Leu He 2 90 2 95 300
Arg Ser Lys Tyr Asn Gin Asn Ser Asn Tyr He Asn Tyr Arg Asn Leu 305 310 315 320
Tyr He Gly Glu Lys Phe He He Arg Arg Glu Ser Asn Ser Gin Ser 325 330 335
He Asn Asp Asp He Val Arg Lys Glu Asp Tyr He His Leu Asp Leu 340 345 350
Val Leu His His Glu Glu Trp Arg Val Tyr Ala Tyr Lys Tyr Ple Lys 355 360 365
Glu Gin Glu Glu Lys Leu Phe Leu Ser He He Ser Asp Ser Asn Glu 370 375 380
Phe Tyr Lys Thr He Glu He Lys Glu Tyr Asp Glu Gin Pro Ser Tyr 385 390 395 400
Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Asp He 405 410 415
Gly Leu He Gly He His Arg Phe Tyr Glu Ser Gly Val Leu Arg Lys 420 425 430
Lyε Tyr Lys Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu Lys Glu Val 435 440 ' ' 445
Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys Asn Trp Gin Phe He 450 ' 455 460
Pro Lys Asp Glu Gly Trp Thr Glu 465 470
(2) INFORMATION FOR SEQ ID NO : 45 :
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1547 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(11 ) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 108..1523 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 45 :
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His
1
CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His He Glu Gly 5 10 15
CGT CAT ATG GCT AGC ATG GCT GAT ACA ATA CTA ATA GAA ATG TTT AAT 212 Arg His Met Ala Ser Met Ala Asp Thr He Leu He Glu Met Phe Asn 20 25 30 35
AAA TAT AAT AGC GAA ATT TTA AAT AAT ATT ATC TTA AAT TTA AGA TAT 260 Lys Tyr Asn Ser Glu He Leu Asn Asn He He Leu Asn Leu Arg Tyr 40 45 50 ΛAG GAT AAT AAT TTA ATA GAT TTA TCA GGA TAT GGG GCA AAG GTA GAG 308 Lys Asp Asn Asn Leu He Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu 55 60 65
GTA TAT GAT GGA GTC GAG CTT AAT GAT AAA AAT CAA TTT AAA TTA ACT 356 Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn Gin Phe Lys Leu Thr 70 75 80
AGT TCA GCA AAT AGT AAG ATT AGA GTG ACT CAA AAT CAG AAT ATC ATA 404 Ser Ser Ala Asn Ser Lys He Arg Val Thr Gin Asn Gin Asn He He 85 90 95
TTT AAT AGT GTG TTC CTT GAT TTT AGC GTT AGC TTT TGG ATA AGA ATA 4S2 Pne Asn Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp He Arg He 100 105 110 115
CCT AAA TAT AAG AAT GAT GGT ATA CAA AAT TAT ATT CAT AAT GAA TAT 500 Pro Lyε Tyr Lys Asn Asp Gly He Gin Asn Tyr He His Asn Glu Tyr
120 125 130 ACA ATA ATT AAT TGT ATG AAA AAT AAT TCG GGC TGG AAA ATA TCT ATT 548 Thr He He Asn Cys Met Lys Asn Asn Ser Gly Trp Lys He Ser He 135 140 145
AGG GGT AAT AGG ATA ATA TGG ACT TTA ATT GAT ATA AAT GGA AAA ACC 596 Arg Gly Asn Arg He He Trp Thr Leu He Asp He Asn Gly Lys Thr 150 155 160
AAA TCG GTA TTT TTT GAA TAT AAC ATA AGA GAA GAT ATA TCA GAG TAT 644 Lys Ser Val Phe Phe Glu Tyr Asn He Arg Glu Asp He Ser Glu Tyr 165 170 175
ATA AAT AGA TGG TTT TTT GTA ACT ATT ACT AAT AAT TTG AAT AAC GCT 692
He Asn Arg Trp Phe Phe Val Thr He Thr Asn Asn Leu Asn Asn Ala
180 185 190 195
AAA ATT TAT ATT AAT GGT AAG CTA GAA TCA AAT ACA GAT ATT AAA GAT 740
Lys He Tyr He Asn Gly Lys Leu Glu Ser Asn Thr Asp He Lys Asp
200 205 210 ATA AGA GAA GTT ATT GCT AAT GGT GAA ATA ATA TTT AAA TTA GAT GGT 788 He A.rg Glu Val He Ala Asn Gly Glu He He Phe Lys Leu Asp Gly 215 220 225
GAT ATA GAT AGA ACA CAA TTT ATT TGG ATG AAA TAT TTC AGT ATT TTT 836 Asp He Asp Arg Thr Gin Phe He Trp Met Lys Tyr Phe Ser He Phe 230 235 240
AAT ACG GAA TTA AGT CAA TCA AAT ATT GAA GAA AGA TAT AAA ATT CAA 884 Asn Thr Glu Leu Ser Gin Ser Asn He Glu Glu Arg Tyr Lys He Gin 245 250 255
TCA TAT AGC GAA TAT TTA AAA GAT TTT TGG GGA AAT CCT TTA ATG TAC 932
Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr
260 265 270 275
AAT AAA GAA TAT TAT ATG TTT AAT GCG GGG AAT AAA AAT TCA TAT ATT 980
Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr He 280 285 290 AAA CTA AAG AAA GAT TCA CCT GTA GGT GAA ATT TTA ACA CGT AGC AAA 1028 Lyε Leu Lys Lyε Asp Ser Pro Val Gly Glu He Leu Thr Arg Ser Lys 295 300 305
TAT AAT CAA AAT TCT AAA TAT ATA AAT TAT AGA GAT TTA TAT ATT GGA 1076 Tyr Asn Gin Asn Ser Lys Tyr He Asn Tyr Arg Asp Leu Tyr He Gly 310 315 320 GAA AAA TTT ATT ATA AGA AGA AAG TCA AAT TCT CAA TCT ATA AAT GAT 1124
Glu Lys Phe He He Arg Arg Lys Ser Asn Ser Gin Ser He Asn Asp 325 330 335 GAT ATA GTT AGA AAA GAA GAT TAT ATA TAT CTA GAT TTT TTT AAT TTA 1172
Asp He Val Arg Lys Glu Asp Tyr He Tyr Leu Asp Phe Phe Asn Leu
340 345 ' 350 355
AAT CAA GAG TGG AGA GTA TAT ACC TAT AAA TAT TTT AAG AAA GAG GAA 1220 Asn Gin Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lyε Lys Glu Glu
360 365 370
GAA AAA TTG TTT TTA GCT CCT ATA AGT GAT TCT GAT GAG TTT TAC AAT 1268
Glu Lys Leu Phe Leu Ala Pro He Ser Asp Ser Asp Glu Phe Tyr Asn 375 380 385
ACT ATA CAA ATA AAA GAA TAT GAT GAA CAG CCA ACA TAT AGT TGT CAG 1316
Thr He Gin He Lys Glu Tyr Asp Glu Gin Pro Thr Tyr Ser Cys Gin 390 395 400
TTG CTT TTT AAA AAA GAT GAA GAA AGT ACT GAT GAG ATA GGA TTG ATT 1364
Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu He Gly Leu He 405 ' 410 ' 415 GGT ATT CAT CGT TTC TAC GAA TCT GGA ATT GTA TTT GAA GAG TAT AAA 1412
Gly He His Arg Phe Tyr Glu Ser Gly He Val Phe Glu Glu Tyr Lys
420 425 430 435
GAT TAT TTT TGT ATA AGT AAA TGG TAC TTA AAA GAG GTA AAA AGG AAA 1460 Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys
440 445 450
CCA TAT AAT TTA AAA TTG GGA TGT AAT TGG CAG TTT ATT CCT AAA GAT 1508
Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp Gin Phe He Pro Lys Asp " 455 460 465
GAA GGG TGG ACT GAA TAAAAGCTTG CGGCCGCACT CGAG 1547
Glu Gly Trp Thr Glu 470
(2) INFORMATION FOR SEQ ID NO : 46
(l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 472 ammo acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: protein (χι) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 He Glu Gly Arg His Met Ala Ser Met Ala Asp Thr He Leu He Glu
20 25 30
Met Phe Asn Lys Tyr Asn Ser Glu He Leu Asn Asn He He Leu Asn 35 40 45
Leu Arg Tyr Lys Asp Asn Asn Leu He Asp Leu Ser Gly Tyr Gly Ala 50 55 60
Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn Gin Phe 65 70 75 80
Lys Leu Thr Ser Ser Ala Asn Ser Lys He Arg Val Thr Gin Asn Gin
85 " 90 95 Asn He He Phe Asn Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp 100 105 110
He Arg He Pro Lys Tyr Lys Asn Asp Gly He Gin Asn Tyr He His 115 120 125
Asn Glu Tyr Thr He He Asn Cys Met Lys Asn Asn Ser Gly Trp Lvs 130 135 140
He Ser He Arg Gly Asn Arg He He Trp Thr Leu He Asp He Asn 145 150 155 160
Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn He Arg Glu Asp He 165 170 175 Ser Glu Tyr He Asn Arg Trp Phe Phe Val Thr He Thr Asn Asn Leu
180 185 190
Asn Asn Ala Lys He Tyr He Asn Gly Lys Leu Glu Ser Asn Thr Asp 195 200 205
He Lys Asp He Arg Glu Val He Ala Asn Gly Glu He He Phe Lyε 210 215 220
Leu Asp Gly Asp He Asp Arg Thr Gin Phe He Trp Met Lys Tyr Phe
225 230 235 240
Ser He Phe Asn Thr Glu Leu Ser Gin Ser Asn He Glu Glu Arg Tyr 245 250 255
Lys He Gin Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro 260 265 270
Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn 275 280 285
Ser Tyr He Lys Leu Lys Lys Asp Ser Pro Val Gly Glu He Leu Thr 290 295 300
Arg Ser Lys Tyr Asn Gin Asn Ser Lys Tyr He Asn Tyr Arg Asp Leu 305 ' 310 315 ' 320
Tyr He Gly Glu Lys Phe He He Arg Arg Lys Ser Asn Ser Gin Ser
325 330 335 He Asn Asp Asp He Val Arg Lys Glu Asp Tyr He Tyr Leu Asp Phe
340 345 350
Phe Asn Leu Asn Gin Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys 355 360 365
Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro He Ser Asp Ser Asp Glu 370 375 380
Phe Tyr Asn Thr He Gin He Lys Glu Tyr Asp Glu Gin Pro Thr Tyr 385 390 395 400
Ser Cys Gin Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu He 405 410 415 Gly Leu He Gly He His Arg Phe Tyr Glu Ser Gly He Val Phe Glu
420 425 430
Glu Tyr Lys Asp Tyr Phe Cys He Ser Lys Trp Tyr Leu Lys Glu Val 435 ' 440 445
Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn Trp Gin Phe He 450 455 460
Pro Lys Asp Glu Gly Trp Thr Glu 465 470 (2) INFORMATION FOR SEQ ID NO 47
U) SEQUENCE CHARACTERISTICS
(A) LENGTH 31 base pairs (B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY lineal
Ui ) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "DNA"
(xi) SEQUENCE DESCRIPTION SEQ ID NO 47
CGCCATGGCT GATACAATAC TAATAGAAAT G 31
(2) INFORMATION FOR SEQ ID NO 48
U) SEQUENCE CHARACTERISTICS
(A) LENGTH 29 base pairs (B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
..1) MOLECULE TYPE other nucleic acid i A ) DESCRIPTION /desc = 'DNA'
(/l) SEQUENCE DESCRIPTION SEQ ID NO 48
GCAAGCTTTT ATTCAGTCCA CCCTTCATC 29
(2) INFORMATION FOR SEQ ID NO 49 r) SEQUENCE CHARACTERISTICS
(A) LENGTH 3753 base pair- (B) TYPE nucleic acid
(C) STRANDEDNESS double
(D) TOPOLOGY linear
Ui) MOLECULE TYPE DNA (genomic)
(ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 3750 r ± ) SEQUENCE DESCRIPTION SEQ ID NO 49
ATG CCA ACA ATT AAT AGT TTT AAT TAT AAT GAT CCT GTT AAT AAT AGA 48
Met Pro Thr He Asn Sei Phe Asn Tyr Asn Asp Pro Val Asn Asn Aig
1 5 10 15
ACA ATT TTA TAT ATT AAA CCA GGC GGT TGT CAA CAA TTT TAT AAA TCA 96
Thr He Leu Tyr He Lys Pro Gly Gly Cys Gin Gin Phe Tyr Lvs Sei
20 25 30 TTT AAT ATT ATG AAA AAT ATT TGG ATA ATT CCA GAG AGA AAT GTA ATT 144
Phe Asn He Met Lys Asn He Trp He He Pro Glu Arg Asn Val He
35 40 45
GGT ACA ATT CCC CAA GAT TTT CTT CCG CCT ACT TCA TTG AAA AAT GGA 192 Gly Thr He Pro Gin Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly
50 55 60
GAT AGT AGT TAT TAT GAC CCT AAT TAT TTA CAA AGT GAT CAA GAA AAG 240
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gin Ser Asp Gin Glu Lvs 65 70 " 75 80
GAT AAA TTT TTA AAA ATA GTC ACA AAA ATA TTT AAT AGA ATA AAT GAT 288
Asp Lys Phe Leu Lys He Val Thr Lvs He Phe Asn Arg He Asn Asp
85 90 95
507 - AAT CTT TCA GGA AGG ATT TTA TTA GAA GAA CTG TCA AAA GCT AAT CCA 336 Asn Leu Ser Gly Arg He Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 TAT TTA GGA AAT GAT AAT ACT CCA GAT GGT GAC TTC ATT ATT AAT GAT 384 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe He He Asn Asp 115 120 125
GCA TCA GCA GTT CCA ATT CAA TTC TCA AAT GGT AGC CAA AGC ATA CTA 432 Ala Ser Ala Val Pro He Gin Phe Ser Asn Gly Ser Gin Ser He Leu 130 135 140
TTA CCT AAT GTT ATT ATA ATG GGA GCA GAG CCT GAT TTA TTT GAA ACT 480 Leu Pro Asn Val He He Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160
AAC AGT TCC AAT ATT TCT CTA AGA AAT AAT TAT ATG CCA AGC AAT CAC 528
Asn Ser Ser Asn He Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn Hiε 165 170 175
GGT TTT GGA TCA ATA GCT ATA GTA ACA TTC TCA CCT GAA TAT TCT TTT 576
Gly Phe Gly Ser He Ala He Val Thr Phe Ser Pro Glu Tyr Ser Phe
180 185 190 AGA TTT AAA GAT AAT AGT ATG AAT GAA TTT ATT CAA GAT CCT GCT CTT 624 Arg Phe Lys Asp Asn Ser Met Aεn Glu Phe He Gin Asp Pro Ala Leu 195 200 205
ACA TTA ATG CAT GAA TTA ATA CAT TCA TTA CAT GGA CTA TAT GGG GCT 672 Thr Leu Met His Glu Leu He His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220
AAA GGG ATT ACT ACA AAG TAT ACT ATA ACA CAA AAA CAA AAT CCC CTA 720 Lys Gly He Thr Thr Lys Tyr Thr He Thr Gin Lys Gin Asn Pro Leu 225 230 235 240
ATA ACA AAT ATA AGA GGT ACA AAT ATT GAA GAA TTC TTA ACT TTT GGA 768
He Thr Asn He Arg Gly Thr Asn He Glu Glu Phe Leu Thr Phe Gly
245 250 255
GGT ACT GAT TTA AAC ATT ATT ACT AGT GCT CAG TCC AAT GAT ATC TAT 816
Gly Thr Asp Leu Asn He He Thr Ser Ala Gin Ser Asn Asp He Tyr
260 265 270 ACT AAT CTT CTA GCT GAT TAT AAA AAA ATA GCG TCT AAA CTT AGC AAA 864 Thr Asn Leu Leu Ala Asp Tyr Lys Lys He Ala Ser Lys Leu Ser Lyε 275 280 285
GTA CAA GTA TCT AAT CCA CTA CTT AAT CCT TAT AAA GAT GTT TTT GAA 912 Val Gin Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300
GCA AAG TAT GGA TTA GAT AAA GAT GCT AGC GGA ATT TAT TCG GTA AAT 960 Ala Lys Tyr Gly Leu Asp Lys Aεp Ala Ser Gly He Tyr Ser Val Aεn 305 310 315 320
ATA AAC AAA TTT AAT GAT ATT TTT AAA AAA TTA TAC AGC TTT ACG GAA 1008
He Asn Lys Phe Asn Asp He Phe Lys Lys Leu Tyr Ser Phe Thr Glu
325 330 335
TTT GAT TTA GCA ACT AAA TTT CAA GTT AAA TGT AGG CAA ACT TAT ATT 1056
Phe Asp Leu Ala Thr Lys Phe Gin Val Lys Cys Arg Gin Thr Tyr He
340 345 350 GGA CAG TAT AAA TAC TTC AAA CTT TCA AAC TTG TTA AAT GAT TCT ATT 1104 Gly Gin Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser He 355 360 365
TAT AAT ATA TCA GAA GGC TAT AAT ATA AAT AAT TTA AAG GTA AAT TTT 1152 Tyr Asn He Ser Glu Gly Tyr Asn He Asn Asn Leu Lys Val Asn Phe
508 - 370 375 380
AGA GGA CAG AAT GCA AAT TTA AAT CCT AGA ATT ATT ACA CCA ATT ACA 1200 Arg Gly Gin Asn Ala Asn Leu Asn Pro Arg He He Thr Pro He Thr 3B5 390 395 400
GGT AGA GGA CTA GTA AAA AAA ATC ATT AGA TTT TGT AAA AAT ATT GTT 1248 Gly Arg Gly Leu Val Lys Lys He He Arg Phe Cys Lys Asn He Val 405 ' 410 415
TCT GTA AAA GGC ATA AGG AAA TCA ATA TGT ATC GAA ATA AAT AAT GGT 1296 Ser Val Lys Gly He Arg Lys Ser He Cys He Glu He Asn Asn Gly 420 425 430
GAG TTA TTT TTT GTG GCT TCC GAG AAT AGT TAT AAT GAT GAT AAT ATA 1344 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn He 435 440 445
AAT ACT CCT AAA GAA ATT GAC GAT ACA GTA ACT TCA AAT AAT AAT TAT 1392 Asn Thr Pro Lys Glu He Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460
GAA AAT GAT TTA GAT CAG GTT ATT TTA AAT TTT AAT AGT GAA TCA GCA 1440 Glu Asn Asp Leu Asp Gin Val He Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480
CCT GGA CTT TCA GAT GAA AAA TTA AAT TTA ACT ATC CAA AAT GAT GCT 1488 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr He Gin Asn Asp Ala 4B5 490 495
TAT ATA CCA AAA TAT GAT TCT AAT GGA ACA AGT GAT ATA GAA CAA CAT 1536 Tyr He Pro Lys Tyr Asp Ser Aεn Gly Thr Ser Asp He Glu Gin His 500 505 510 GAT GTT AAT GAA CTT AAT GTA TTT TTC TAT TTA GAT GCA CAG AAA GTG 1584 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gin Lys Val 515 520 525
CCC GAA GGT GAA AAT AAT GTC AAT CTC ACC TCT TCA ATT GAT ACA GCA 1632 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser He Asp Thr Ala 530 535 540
TTA TTA GAA CAA CCT AAA ATA TAT ACA TTT TTT TCA TCA GAA TTT ATT 1680 Leu Leu Glu Gin Pro Lys He Tyr Thr Phe Phe Ser Ser Glu Phe He 545 550 555 560
AAT AAT GTC AAT AAA CCT GTG CAA GCA GCA TTA TTT GTA AGC TGG ATA 1728 Asn Asn Val Asn Lys Pro Val Gin Ala Ala Leu Phe Val Ser Trp He 565 570 575
CAA CAA GTA TTA GTA GAT TTT ACT ACT GAA GCT AAC CAA AAA AGT ACT 1776 Gin Gin Val Leu Val Aεp Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr 580 585 590 GTT GAT AAA ATT GCA GAT ATT TCT ATA GTT GTT CCA TAT ATA GGT CTT 1824 Val Asp Lys He Ala Asp He Ser He Val Val Pro Tyr He Gly Leu 595 600 605
GCT TTA AAT ATA GGA AAT GAA GCA CAA AAA GGA AAT TTT AAA GAT GCA 1872 Ala Leu Asn He Gly Asn Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala 610 615 620
CTT GAA TTA TTA GGA GCA GGT ATT TTA TTA GAA TTT GAA CCC GAG CTT 1920 Leu Glu Leu Leu Gly Ala Gly He Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640
TTA ATT CCT ACA ATT TTA GTA TTC ACG ATA AAA TCT TTT TTA GGT TCA 1968 Leu He Pro Thr He Leu Val Phe Thr He Lys Ser Phe Leu Gly Ser 645 650 655 TCT GAT AAT AAA AAT AAA GTT ATT AAA GCA ATA AAT AAT GCA TTG AAA 2016 Ser Asp Asn Lys Asn Lys Val He Lys Ala He Asn Asn Ala Leu Lys 660 665 670 GAA AGA GAT GAA AAA TGG AAA GAA GTA TAT AGT TTT ATA GTA TCG AAT 2064 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe He Val Ser Asn 675 680 685
TGG ATG ACT AAA ATT AAT ACA CAA TTT AAT AAA AGA AAA GAA CAA ATG 2112 Trp Met Thr Lys He Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met 690 695 700
TAT CAA GCT TTA CAA AAT CAA GTA AAT GCA CTT AAA GCA ATA ATA GAA 2160 Tyr Gin Ala Leu Gin Asn Gin Val Asn Ala Leu Lys Ala He He Glu 705 710 715 720
TCT AAG TAT AAT AGT TAT ACT TTA GAA GAA AAA AAT GAG CTT ACA AAT 2208
Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn
725 730 735
AAA TAT GAT ATT GAG CAA ATA GAA AAT GAA CTT AAT CAA AAG GTT TCT 2256
Lys Tyr Asp He Glu Gin He Glu Asn Glu Leu Asn Gin Lys Val Ser
740 745 750 ATA GCA ATG AAT AAT ATA GAC AGG TTC TTA ACT GAA AGT TCT ATA TCT 2304 He Ala Met Asn Asn He Aεp Arg Phe Leu Thr Glu Ser Ser He Ser 755 760 765
TAT TTA ATG AAA TTA ATA AAT GAA GTA AAA ATT AAT AAA TTA AGA GAA 2352 Tyr Leu Met Lys Leu He Asn Glu Val Lys He Asn Lys Leu Arg Glu 770 ' 775 780
TAT GAT GAA AAT GTT AAA ACG TAT TTA TTA GAT TAT ATT ATA AAA CAT 2400 Tyr Asp Glu Aεn Val Lys Thr Tyr Leu Leu Asp Tyr He He Lys His 785 790 ' 795 800
GGA TCA ATC TTG GGA GAG AGT CAG CAA GAA CTA AAT TCT ATG GTA ATT 2448
Gly Ser He Leu Gly Glu Ser Gin Gin Glu Leu Asn Ser Met Val He 805 810 815
GAT ACC CTA AAT AAT AGT ATT CCT TTT AAG CTT TCT TCT TAT ACA GAT 2496
Asp Thr Leu Asn Asn Ser He Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 GAT AAA ATT TTA ATT TCA TAT TTT AAT AAG TTC TTT AAG AGA ATT AAA 2544 Asp Lys He Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lys 835 " 840 845
AGT AGT TCT GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT 2592 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860
ACT TCA GGA TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA 2640 Thr Ser Gly Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys 865 870 875 880
TAT CCA ACT AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT 2688 Tyr Pro Thr Asn Lys Asn Gin Phe Gly He Tyr Asn Aεp Lys Leu Ser 885 890 895
GAA GTT AAT ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT 2736 Glu Val Asn He Ser Gin Asn Aεp Tyr He He Tyr Asp Asn Lys Tyr 900 905 910 AAA AAT TTT AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT 2784
Lys Asn Phe Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn 915 920 925
AAG ATA GTA AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGG 2832 Lys He Val Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg 930 935 940
GAT AAT AAT TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT 2880
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He 945 950 955 960
TGG ACA TTG CAA GAT AAT TCA GGA ATT AAT CAA AAA TTA GCA TTT AAC 2928
Trp Thr Leu Gin Asp Asn Ser Gly He Asn Gin Lys Leu Ala Phe Asn
965 970 975
TAT GGT AAC GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT 2976
Tyr Gly Asn Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe 980 985 990 GTA ACT ATA ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT 3024
Val Thr He Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn 995 1000 1005
GGA AAT TTA ATA GAT AAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT 3072 Gly Asn Leu He Asp Lys Lys Ser He Leu Asn Leu Gly Asn He His 1010 1015 1020
GTT AGT GAC AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA 3120
Val Ser Asp Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040
TAT ATT GGT ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA 3168
Tyr He Gly He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu
1045 1050 1055
ACA GAA ATT CAA ACT TTA TAT AAC AAT GAA CCT AAT GCA AAT ATT TTA 3216
Thr Glu He Gin Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn He Leu 1060 1065 1070 AAG GAT TTT TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA 3264
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085
TTA AAT GTG TTA AAA CCA AAT AAC TTT ATT AAT AGG AGA ACA GAT TCT 3312 Leu Aεn Val Leu Lys Pro Asn Asn Phe He Asn Arg Arg Thr Asp Ser 1090 1095 1100
ACT TTA AGC ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA 3360
Thr Leu Ser He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg 1105 1110 1115 1120
TTA TAT AGT GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT 3408
Leu Tyr Ser Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser
1125 1130 1135
ACT AAC GAT AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT 3456
Thr Aεn Aεp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe 1140 1145 1150 GTA GCC AGC AAA ACT CAC TTA CTT CCA TTA TAT GCT GAT ACA GCT ACC 3504
Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165
ACA AAT AAA GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT 3552 Thr Asn Lys Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe 1170 ■ 1175 1180
AAT CAA GTA GTA GTT ATG AAT TCA GTA GGA TGT ACA ATG AAT TTT AAA 3600
Asn Gin Val Val Val Met Asn Ser Val Gly Cys Thr Met Asn Phe Lys 1185 1190 1195 1200 AAT AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT 3648 Asn Asn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 1210 ' ' 1215 GTA GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT AAT ACA AAC 3696 Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn 1220 1225 1230
AGC AAT GGA TTT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA 3744 Ser Asn Gly Phe Phe Trp Asn Phe He Ser Glu Glu His Gly Trp Gin 1235 1240 1245
GAA AAA TAA 3753
Glu Lys 1 50
(2) INFORMATION FOR SEQ ID NO: 50:
U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1250 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
Ui) MOLECULE TYPE: protem
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
Met Pro Thr He Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asn Arg
1 5 10 15
Thr He Leu Tyr He Lys Pro Gly Gly Cys Gin Gin Phe Tyr Lys Ser 20 25 30
Phe Asn He Met Lys Asn He Trp He He Pro Glu Arg Asn Val He 35 40 45
Gly Thr He Pro Gin Aεp Phe Leu Pro Pro Thr Ser Leu Lys Aεn Gly 50 55 60 Aεp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gin Ser Asp Gin Glu Lys 65 70 75 80
Asp Lys Phe Leu Lys He Val Thr Lys He Phe Asn Arg He Asn Asp 85 90 95
Asn Leu Ser Gly Arg He Leu Leu Glu Glu Leu Ser Lyε Ala Asn Pro 100 105 110
Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe He He Asn Asp 115 120 125
Ala Ser Ala Val Pro He Gin Phe Ser Asn Gly Ser Gin Ser He Leu
130 135 140
Leu Pro Asn Val He He Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160
Asn Ser Ser Asn He Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175
Gly Phe Gly Ser He Ala He Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190
Arg Phe Lys Asp Asn Ser Met Asn Glu Phe He Gin Asp Pro Ala Leu 195 200 205
Thr Leu Met His Glu Leu He His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys Gly He Thr Thr Lys Tyr Thr He Thr Gin Lys Gin Asn Pro Leu 225 230 235 240
He Thr Asn He Arg Gly Thr Asn He Glu Glu Phe Leu Thr Phe Gly 245 250 255
Gly Thr Asp Leu Asn He He Thr Ser Ala Gin Ser Asn Asp He Tyr 260 265 270
Thr Asn Leu Leu Ala Asp Tyr Lys Lys He Ala Ser Lys Leu Ser Lys 275 280 285
Val Gin Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Rhe Glu 290 295 300
Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly He Tyr Ser Val Asn 305 310 315 320
He Asn Lys Phe Asn Asp He Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335
Phe Asp Leu Ala Thr Lys Phe Gin Val Lys Cys Arg Gin Thr Tyr He 340 345 350
Gly Gin Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser He ' 355 360 365
Tyr Asn He Ser Glu Gly Tyr Asn He Asn Aεn Leu Lyε Val Asn Phe 370 375 380 Arg Gly Gin Asn Ala Asn Leu Asn Pro Arg He He Thr Pro He Thr 385 390 395 400
Gly Arg Gly Leu Val Lys Lys He He Arg Phe Cys Lys Aεn He Val 405 410 415
Ser Val Lys Gly He Arg Lys Ser He Cys He Glu He Aεn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn He 435 440 445
Asn Thr Pro Lys Glu He Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn Aεp Leu Asp Gin Val He Leu Asn Phe Asn Ser Glu Ser Ala
465 470 475 480
Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr He Gin Asn Asp Ala 485 490 495
Tyr He Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp He Glu Gin His 500 505 510
Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gin Lys Val 515 520 525
Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser He Asp Thr Ala
530 535 540 Leu Leu Glu Gin Pro Lys He Tyr Thr Phe Phe Ser Ser Glu Phe He
545 550 ' 555 560
Asn Asn Val Asn Lys Pro Val Gin Ala Ala Leu Phe Val Ser Trp He
565 570 575
Gin Gin Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr
580 585 590
Val Λεp Lys He Ala Asp He Ser He Val Val Pro Tyr He Gly Leu * 595 600 605 Ala Leu Asn He Gly Asn Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala 610 615 620
Leu Glu Leu Leu Gly Ala Gly He Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640
Leu He Pro Thr He Leu Val Phe Thr He Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val He Lys Ala He Asn Asn Ala Leu Lys
660 665 670
Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe He Val Ser Asn 675 680 685
Trp Met Thr Lys He Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met 690 695 700
Tyr Gin Ala Leu Gin Asn Gin Val Asn Ala Leu Lys Ala He He Glu 705 710 715 720
Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp He Glu Gin He Glu Asn Glu Leu Asn Gin Lys Val Ser
740 745 750
He Ala Met Asn Asn He Asp Arg Phe Leu Thr Glu Ser Ser He Ser 755 760 765
Tyr Leu Met Lys Leu He Asn Glu Val Lys He Asn Lys Leu Arg Glu 770 775 780
Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asp Tyr He He Lyε Hiε 785 790 795 800
Gly Ser He Leu Gly Glu Ser Gin Gin Glu Leu Asn Ser Met Val He 805 810 815 Asp Thr Leu Asn Asn Ser He Pro Phe Lys Leu Ser Ser Tyr Thr Asp
820 825 830
Asp Lys He Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lys
835 840 845
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp
850 855 860
Thr Ser Gly Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys 865 870 875 880
Tyr Pro Thr Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn He Ser Gin Asn Asp Tyr He He Tyr Aεp Asn Lys Tyr
900 905 910
Lys Asn Phe Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn 915 920 925
Lys He Val Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg 930 935 940
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He 945 950 " 955 960
Trp Thr Leu Gin Asp Asn Ser Gly He Asn Gin Lys Leu Ala Phe Asn
965 970 975 Tyr Gly Asn Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe 980 985 990
Val Thr He Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn 995 1000 1005
Gly Asn Leu He Asp Lys Lys Ser He Leu Asn Leu Gly Asn He His 1010 1015 1020
Val Ser Asp Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 1040
Tyr He Gly He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr Glu He Gin Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn He Leu
1060 1065 1070
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085
Leu Asn Val Leu Lys Pro Asn Asn Phe He Asn Arg Arg Thr Asp Ser 1090 1095 1100
Thr Leu Ser He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg 1105 1110 1115 1120
Leu Tyr Ser Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser 1125 ' 1130 1135
Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe 1140 1145 1150
Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165
Thr Asn Lys Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180
Asn Gin Val Val Val Met Asn Ser Val Gly Cys Thr Met Asn Phe Lys 1185 1190 1195 1200
Asn Aεn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 1210 1215
Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr 1220 1225 1230
Ser Asn Gly Phe Phe Trp Asn Phe He Ser Glu Glu His Gly Trp Gin 1235 1240 1245
Glu Lys 1250
(2) INFORMATION FOR SEQ ID NO: 51:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3759 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
Ui) MOLECULE TYPE: DNA (genomic)
( ix ) FEATURE : (A) NAME/KEY: CDS
(B) LOCATION: 1..3756
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: ATG CCA AAA ATT AAT AGT TTT AAT TAT AAT GAT CCT GTT AAT GAT AGA 48 Met Pro Lys He Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg
1 5 10 15
ACA ATT TTA TAT ATT AAA CCA GGC GGT TGT CAA GAA TTT TAT AAA TCA 96
5 Thr He Leu Tyr He Lys Pro Gly Gly Cys Gin Glu Phe Tyr Lys Ser
20 25 30
TTT AAT ATT ATG AAA AAT ATT TGG ATA ATT CCA GAG AGA AAT GTA ATT 144
Phe Asn He Met Lys Asn He Trp He He Pro Glu Arg Asn Val He
10 35 40 45
GGT ACA ACC CCC CAA GAT TTT CAT CCG CCT ACT TCA TTA AAA AAT GGA 192
Gly Thr Thr Pro Gin Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly
50 55 60 15
GAT AGT AGT TAT TAT GAC CCT AAT TAT TTA CAA AGT GAT GAA GAA AAG 240
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gin Ser Asp Glu Glu Lys
65 70 75 80
20 GAT AGA TTT TTA AAA ATA GTC ACA AAA ATA TTT AAT AGA ATA AAT AAT 288 Asp Arg Phe Leu Lys He Val Thr Lys He Phe Asn Arg He Asn Asn 85 90 95
AAT CTT TCA GGA GGG ATT TTA TTA GAA GAA CTG TCA AAA GCT AAT CCA 336 25 Asn Leu Ser Gly Gly He Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro
100 105 110
TAT TTA GGG AAT GAT AAT ACT CCA GAT AAT CAA TTC CAT ATT GGT GAT 384 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gin Phe His He Gly Asp 30 115 120 125
GCA TCA GCA GTT GAG ATT AAA TTC TCA AAT GGT AGC CAA GAC ATA CTA 432
Ala Ser Ala Val Glu He Lys Phe Ser Asn Gly Ser Gin Asp He Leu
130 135 140
3.3
TTA CCT AAT GTT ATT ATA ATG GGA GCA GAG CCT GAT TTA TTT GAA ACT 480 Leu Pro Asn Val He He Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160
40 AAC AGT TCC AAT ATT TCT CTA AGA AAT AAT TAT ATG CCA AGC AAT CAC 528 Asn Ser Ser Asn He Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175
GGT TTT GGA TCA ATA GCT ATA GTA ACA TTC TCA CCT GAA TAT TCT TTT 576 45 ly Phe Gly Ser He Ala He Val Thr Phe Ser Pro Glu Tyr Ser Phe
180 185 190
AGA TTT AAT GAT AAT AGT ATG AAT GAA TTT ATT CAA GAT CCT GCT CTT 624 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe He Gin Asp Pro Ala Leu 50 195 200 205
ACA TTA ATG CAT GAA TTA ATA CAT TCA TTA CAT GGA CTA TAT GGG GCT 672 Thr Leu Met His Glu Leu He His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220
55
AAA GGG ATT ACT ACA AAG TAT ACT ATA ACA CAA AAA CAA AAT CCC CTA 720 Lys Gly He Thr Thr Lys Tyr Thr He Thr Gin Lys Gin Asn Pro Leu 225 230 235 240
60 ATA ACA AAT ATA AGA GGT ACA AAT ATT GAA GAA TTC TTA ACT TTT GGA 768 He Thr Asn He Arg Gly Thr Asn He Glu Glu Phe Leu Thr Phe Gly 245 ' 250 255
GGT ACT GAT TTA AAC ATT ATT ACT AGT GCT CAG TCC AAT GAT ATC TAT 816 65 Gly Thr Asp Leu Asn He He Thr Ser Ala Gin Ser Asn Asp He Tyr
260 265 270
ACT AAT CTT CTA GCT GAT TAT AAA AAA ATA GCG TCT AAA CTT AGC AAA 864 Thr Asn Leu Leu Ala Asp Tyr Lys Lys He Ala Ser Lys Leu Ser Lys 70 275 280 285 GTA CAA GTA TCT AAT CCA CTA CTT AAT CCT TAT AAA GAT GTT TTT GAA 912
Val Gin Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 ' 300 GCA AAG TAT GGA TTA GAT AAA GAT GCT AGC GGA ATT TAT TCG GTA AAT 960
Ala Lvs Tyr Gly Leu Asp Lys Asp Ala Ser Gly He Tyr Ser Val Asn
305 310 315 320
ATA AAC AAA TTT AAT GAT ATT TTT AAA AAA TTA TAC AGC TTT ACG GAA 1008 He Asn Lys Phe Asn Asp He Phe Lys Lys Leu Tyr Ser Phe Thr Glu
325 330 335
TTT GAT TTA GCA ACT AAA TTT CAA GTT AAA TGT AGG CAA ACT TAT ATT 1056
Phe Asp Leu Ala Thr Lys Phe Gin Val Lys Cys Arg Gin Thr Tyr He 340 345 350
GGA CAG TAT AAA TAC TTC AAA CTT TCA AAC TTG TTA AAT GAT TCT ATT 1104
Gly Gin Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser He 355 360 365
TAT AAT ATA TCA GAA GGC TAT AAT ATA AAT AAT TTA AAG GTA AAT TTT 1152
Tyr Asn He Ser Glu Gly Tyr Asn He Asn Asn Leu Lys Val Asn Phe 370 375 380 AGA GGA CAG AAT GCA AAT TTA AAT CCT AGA ATT ATT ACA CCA ATT ACA 1200
Arg Gly Gin Asn Ala Asn Leu Asn Pro Arg He He Thr Pro He Thr
385 ' 390 395 400
GGT AGA GGA CTA GTA AAA AAA ATC ATT AGA TTT TGT AAA AAT ATT GTT 1248 Gly Arg Gly Leu Val Lys Lys He He Arg Phe Cys Lys Asn He Val
405 410 415
TCT GTA AAA GGC ATA AGG AAA TCA ATA TGT ATC GAA ATA AAT AAT GGT 1296
Ser Val Lys Gly He Arg Lys Ser He Cys He Glu He Asn Asn Gly 420 425 430
GAG TTA TTT TTT GTG GCT TCC GAG AAT AGT TAT AAT GAT GAT AAT ATA 1344
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn He 435 440 ' 445
AAT ACT CCT AAA GAA ATT GAC GAT ACA GTA ACT TCA AAT AAT AAT TAT 1392
Asn Thr Pro Lys Glu He Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 GAA AAT GAT TTA GAT CAG GTT ATT TTA AAT TTT AAT AGT GAA TCA GCA 1440
Glu Asn Asp Leu Asp Gin Val He Leu Asn Phe Asn Ser Glu Ser Ala
465 470 475 480
CCT GGA CTT TCA GAT GAA AAA TTA AAT TTA ACT ATC CAA AAT GAT GCT 1488 Pro Gl/ Leu Ser Asp Glu Lys Leu Aεn Leu Thr He Gin Asn Asp Ala
485 490 495
TAT ATA CCA AAA TAT GAT TCT AAT GGA ACA AGT GAT ATA GAA CAA CAT 1536
Tyr He Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp He Glu Gin His 500 505 510
GAT GTT AAT GAA CTT AAT GTA TTT TTC TAT TTA GAT GCA CAG AAA GTG 1584
Asp Val Asn Glu Leu Aεn Val Phe Phe Tyr Leu Asp Ala Gin Lys Val 515 520 525
CCC GAA GGT GAA AAT AAT GTC AAT CTC ACC TCT TCA ATT GAT ACA GCA 1632
Pro Glu Glv Glu Asn Asn Val Asn Leu Thr Ser Ser He Asp Thr Ala 530 535 540 TTA TTA GAA CAA CCT AAA ATA TAT ACA TTT TTT TCA TCA GAA TTT ATT 1680
Leu Leu Glu Gin Pro Lys He Tyr Thr Phe Phe Ser Ser Glu Phe He
545 550 * 555 560
AAT AAT GTC AAT AAA CCT GTG CAA GCA GCA TTA TTT GTA AGC TGG ATA 1728 Asn Asn Val Asn Lys Pro Val Gin Ala Ala Leu Phe Val Ser Trp He 565 570 575
CAA CAA GTG TTA GTA GAT TTT ACT ACT GAA GCT AAC CAA AAA AGT ACT 1776 Gin Gin Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr 580 585 590
GTT GAT AAA ATT GCA GAT ATT TCT ATA GTT GTT CCA TAT ATA GGT CTT 1824 Val Asp Lys He Ala Asp He Ser He Val Val Pro Tyr He Gly Leu 595 600 605
GCT TTA AAT ATA GGA AAT GAA GCA CAA AAA GGA AAT TTT AAA GAT GCA 1872 Ala Leu Asn He Gly Asn Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala 610 615 620
CTT GAA TTA TTA GGA GCA GGT ATT TTA TTA GAA TTT GAA CCC GAG CTT 1920 Leu Glu Leu Leu Gly Ala Gly He Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640
TTA ATT CCT ACA ATT TTA GTA TTC ACG ATA AAA TCT TTT TTA GGT TCA 1968 Leu He Pro Thr He Leu Val Phe Thr He Lys Ser Phe Leu Gly Ser 645 650 655
TCT GAT AAT AAA AAT AAA GTT ATT AAA GCA ATA AAT AAT GCA TTG AAA 2016 Ser Asp Asn Lys Asn Lys Val He Lys Ala He Asn Asn Ala Leu Lys 660 ' 665 670
GAA AGA GAT GAA AAA TGG AAA GAA GTA TAT AGT TTT ATA GTA TCG AAT 2064 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe He Val Ser Asn 675 680 685
TGG ATG ACT AAA ATT AAT ACA CAA TTT AAT AAA AGA AAA GAA CAA ATG 2112 Trp Met Thr Lys He Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met 690 695 700 TAT CAA GCT TTA CAA AAT CAA GTA AAT GCA ATT AAA ACA ATA ATA GAA 2160 Tyr Gin Ala Leu Gin Asn Gin Val Asn Ala He Lys Thr He He Glu 705 710 715 720
TCT AAG TAT AAT AGT TAT ACT TTA GAG GAA AAA AAT GAG CTT ACA AAT 2208 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn
725 730 735
AAA TAT GAT ATT AAG CAA ATA GAA AAT GAA CTT AAT CAA AAG GTT TCT 2256 Lys Tyi Asp He Lys Gin He Glu Asn Glu Leu Asn Gin Lys Val Ser 740 745 750
ATA GCA ATG AAT AAT ATA GAC AGG TTC TTA ACT GAA AGT TCT ATA TCC 2304
He Ala Met Asn Asn He Asp Arg Phe Leu Thr Glu Ser Ser He Ser 755 760 765
TAT TTA ATG AAA TTA ATA AAT GAA GTA AAA ATT AAT AAA TTA AGA GAA 2352
Tyr Leu Met Lys Leu He Asn Glu Val Lys He Asn Lys Leu Arg Glu 770 775 780 TAT GAT GAG AAT GTC AAA ACG TAT TTA TTG AAT TAT ATT ATA CAA CAT 2400 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr He He Gin His 785 790 795 800
GGA TCA ATC TTG GGA GAG AGT CAG CAA GAA CTA AAT TCT ATG GTA ACT 2448 Gly Ser He Leu Gly Glu Ser Gin Gin Glu Leu Asn Ser Met Val Thr
805 810 815
GAT ACC CTA AAT AAT AGT ATT CCT TTT AAG CTT TCT TCT TAT ACA GAT 2496 Asp Thr Leu Asn Asn Ser He Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830
GAT AAA ATT TTA ATT TCA TAT TTT AAT AAA TTC TTT AAG AGA ATT AAA 2544
Asp Lys He Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lys 835 840 845 AGT AGT TCA GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT 2592
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860
ACT TCA GGA TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA 2640
Thr Ser Gly Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys
865 870 875 880
TAT CCA ACT AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT 2688
Tyr Pro Thr Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lys Leu Ser 885 890 895
GAA GTT AAT ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT 2736
Glu Val Asn He Ser Gin Asn Asp Tyr He He Tyr Asp Asn Lys Tyr 900 905 910
AAA AAT TTT AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT 2784
Lys Asn Phe Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn 915 920 925
AAG ATA GTA AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGA 2832
Lys He Val Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg 930 935 940 GAT AAT AAT TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT 2880
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He
945 950 955 960
TGG ACA TTG CAA GAT AAT GCA GGA ATT AAT CAA AAA TTA GCA TTT AAC 2928 Trp Thr Leu Gin Asp Asn Ala Gly He Asn Gin Lys Leu Ala Phe Asn
965 970 975
TAT GGT AAC GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT 2976
Tyr Gly Asn Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe 980 985 ' 990
GTA ACT ATA ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT 3024
Val Thr He Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn 995 1000 1005
GGA AAT TTA ATA GAT CAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT 3072
Gly Asn Leu He Asp Gin Lys Ser He Leu Asn Leu Gly Asn He His 1010 1015 1020 GTT AGT GAC AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA 3120
Val Ser Asp Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg
1025 1030 1035 ' 1040
TAT ATT GGT ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA 3168 Tyr He Gly He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu
1045 1050 1055
ACA GAA ATT CAA ACT TTA TAT AGC AAT GAA CCT AAT ACA AAT ATT TTG 3216
Thr Glu He Gin Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn He Leu 1060 1065 1070
AAG GAT TTT TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA 3264
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085
TTA AAT GTG TTA AAA CCA AAT AAC TTT ATT GAT AGG AGA AAA GAT TCT 3312 Leu Asn Val Leu Lys Pro Asn Asn Phe He Asp Arg Arg Lys Asp Ser 1090 1095 1100 ACT TTA AGC ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA 3360 Thr Leu Ser He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg 1105 1110 1115 1120
TTA TAT AGT GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT 3408 Leu Tyr Ser Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser 1125 1130 1135
ACT AAC GAT AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT 3456 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe 1140 1145 1150
GTA GCC AGC AAA ACT CAC TTA TTT CCA TTA TAT GCT GAT ACA GCT ACC 3504 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165
ACA AAT AAA GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT 3552 Thr Asn Lys Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 AAT CAA GTA GTA GTT ATG AAT TCA GTA GGA AAT AAT TGT ACA ATG AAT 3600 Asn Gin Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200
TTT AAA AAT AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA 3648 Phe Lys Asn Asn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala
1205 1210 1215
GAT ACT GTA GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT CAT 3696 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230
ACA AAC AGC AAT GGA TGT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA 3744 Thr Asn Ser Asn Gly Cys Phe Trp Aεn Phe He Ser Glu Glu Hiε Gly 1235 1240 1245
TGG CAA GAA AAA TAA 3759
Trp Gin Glu Lys 1250 (?) INFORMATION FOR SEQ ID NO: 52-
(l) SEQUENCE CHARACTE ISTICS:
(A) LENGTH: 1252 ammo acids
(B) TYPE: ammo acid (D) TOPOLOGY: linear
Ui ) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Met Pro Lvs He Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg
1 ' 5 10 15
Thr He Leu Tyr He Lys Pro Gly Gly Cys Gin Glu Phe Tyr Lys Ser 20 25 30
Phe Asn He Met Lys Asn He Trp He He Pro Glu Arg Asn Val He
35 40 45
Gly Thr Thr Pro Gin Asp Phe Hrs Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gin Ser Asp Glu Glu Lys
65 70 75 80
Asp Arg Phe Leu Lys He Val Thr Lys He Phe Asn Arg He Asn Asn
85 90 95
Asn Leu Ser Gly Gly He Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110
Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gin Phe His He Gly Aεp 115 120 125 Ala Ser Ala Val Glu He Lys Phe Ser Asn Gly Ser Gin Asp He Leu 130 135 140
Leu Pro Asn Val He He Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160
Asn Ser Ser Asn He Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175
Gly Phe Gly Ser He Ala He Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190
Arg Phe Asn Asp Asn Ser Met Asn Glu Phe He Gin Asp Pro A a Leu 195 200 205
Thr Leu Met His Glu Leu He His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220
Lys Gly He Thr Thr Lys Tyr Thr He Thr Gin Lys Gin Asn Pro Leu 225 ' 230 235 240
He Thr Asn He Arg Gly Thr Asn He Glu Glu Phe Leu Thr Phe Gly 245 250 255
Gly Tnr Asp Leu Asn He He Thr Ser Ala Gin Ser Asn Asp He Tyr 260 265 270
Thr Asn Leu Leu Ala Asp Tyr Lys Lys He Ala Ser Lys Leu Ser Lyε 275 280 285 Val Gin Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300
Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly He Tyr Ser Val Asn 305 310 315 320
He Asn Lys Phe Asn Asp He Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 ' 330 ' 335
Phe Asp Leu Ala Thr Lys Phe Gin Val Lys Cys Arg Gin Thr Tyr He 340 345 350
Gly Gin Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser He 355 360 365 Tyr Asn He Ser Glu Gly Tyr Asn He Asn Asn Leu Lys Val Asn Phe 370 ' 375 380
Arg Gly Gin Asn Ala Asn Leu Asn Pro Arg He He Thr Pro He Thr 385 390 395 400
Gly Arg Gly Leu Val Lys Lys He He Arg Phe Cys Lys Asn He Val 405 ' 410 415
Ser Val Lys Gly He Arg Lys Ser He Cys He Glu He Asn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Aεp Aεn He 435 440 445 Asn Thr Pro Lys Glu He Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460
Glu Asn Asp Leu Asp Gin Val He Leu Asn Phe Asn Ser Glu Ser Ala
465 470 475 480
Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr He Gin Asn Asp Ala
485 490 495
Tyr He Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp He Glu Gin His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gin Lys Val 515 520 525
Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser He Asp Thr Ala 530 535 540
Leu Leu Glu Gin Pro Lys He Tyr Thr Phe Phe Ser Ser Glu Phe He 545 550 555 560
Asn Asn Val Asn Lys Pro Val Gin Ala Ala Leu Phe Val Ser Trp He 565 570 575
Gin Gin Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gin Lys Ser Thr 580 585 590
Val Asp Lys He Ala Asp He Ser He Val Val Pro Tyr He Gly Leu 595 600 605
Ala Leu Asn He Gly Asn Glu Ala Gin Lys Gly Asn Phe Lys Asp Ala 610 615 620
Leu Glu Leu Leu Gly Ala Gly He Leu Leu Glu Phe Glu Pro Glu Leu
625 630 635 640 Leu He Pro Thr He Leu Val Phe Thr He Lys Ser Phe Leu Gly Ser
645 650 655
Ser Asp Asn Lys Asn Lyε Val He Lys Ala He Asn Asn Ala Leu Lys 660 665 670
Glu Arg Asp Glu Lyε Trp Lys Glu Val Tyr Ser Phe He Val Ser Asn 675 680 685
Trp Met Thr Lys He Asn Thr Gin Phe Asn Lys Arg Lys Glu Gin Met 690 695 700
Tyr Gin Ala Leu Gin Asn Gin Val Asn Ala He Lys Thr He He Glu 705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn
725 ' 730 735
Lys Tyr Asp He Lys Gin He Glu Asn Glu Leu Asn Gin Lys Val Ser
740 745 750
He Ala Met Asn Asn He Asp Arg Phe Leu Thr Glu Ser Ser He Ser
755 760 765
Tyr Leu Met Lys Leu He Asn Glu Val Lys He Asn Lys Leu Arg Glu 770 775 780 '
Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr He He Gin His
785 7 0 795 800 Gly Ser He Leu Gly Glu Ser Gin Gin Glu Leu Asn Ser Met Val Thr
805 810 815
Asp Thr Leu Asn Asn Ser He Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830
Asp Lys He Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lys 835 840 845
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860
Thr Ser Gly Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys 865 870 875 ' ' 880 Tyr Pro Thr Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lys Leu Ser 885 890 895
Glu Val Asn He Ser Gin Asn Asp Tyr He He Tyr Asp Asn Lys Tyr 900 905 910
Lys Asn Phe Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn 915 920 925
Lys He Val Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg 930 935 940
Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He 945 950 955 960 Trp Thr Leu Gin Asp Asn Ala Gly He Asn Gin Lyε Leu Ala Phe Asn
965 970 975
Tyr Gly Asn Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe 980 985 990
Val Thr He Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn 995 1000 1005
Gly Asn Leu He Asp Gin Lys Ser He Leu Asn Leu Gly Asn He His 1010 1015 1020
Val Ser Asp Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg 1025 1030 1035 ' 1040 Tyr He Gly He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu
1045 ' 1050 1055
Thr Glu He Gin Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn He Leu 1060 1065 1070
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085
Leu Asn Val Leu Lys Pro Asn Asn Phe He Asp Arg Arg Lys Asp Ser 1090 ' 1095 1100
Thr Leu Ser He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg 1105 1110 1115 1120 Leu Tyr Ser Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser
1125 1130 1135
Thr Asn Asp Asn Leu Val Arg Lyε Asn Asp Gin Val Tyr He Asn Phe 1140 1145 1150
Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165
Thr Asn Lys Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180
Asn Gin Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 1185 1190 1195 1200 Phe Lys Asn Asn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala
1205 1210 1215
Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220 1225 1230
Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe He Ser Glu Glu His Gly
1235 1240 1245
Trp Gin Glu Lys 1250 (2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTE ISTICS:
(A) LENGTH: 1463 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
Ux) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 108..1460
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60 TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Figure imgf000326_0001
CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His He Glu Gly 5 10 15
CGT CAT ATG GCT AGC ATG GCT CTT TCT TCT TAT ACA GAT GAT AAA ATT 212 Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp Asp Lys He 20 25 30 35
TTA ATT TCA TAT TTT AAT AAG TTC TTT AAG AGA ATT AAA AGT AGT TCT 260
Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lyε Ser Ser Ser 40 45 50
GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT ACT TCA GGA 308
Val Leu Aεn Met Arg Tyr Lyε Asn Asp Lys Tyr Val Asp Thr Ser Gly 55 60 65 TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA TAT CCA ACT 356 Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys Tyr Pro Thr 70 75 80
AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT GAA GTT AAT 404 Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lys Leu Ser Glu Val Asn 85 90 95
ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT AAA AAT TTT 452 He Ser Gin Asn Asp Tyr He He Tyr Asp Asn Lys Tyr Lys Aεn Phe 100 105 110 115
AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT AAG ATA GTA 500
Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn Lys He Val
120 125 130
AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGG GAT AAT AAT 548
Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg Asp Asn Asn
135 140 145 TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT TGG ACA TTG 596 Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He Trp Thr Leu 150 155 160
CAA GAT AAT TCA GGA ATT AAT CAA AAA TTA GCA TTT AAC TAT GGT AAC 644 Gin Asp Asn Ser Gly He Asn Gin Lys Leu Ala Phe Asn Tyr Gly Asn 165 170 175
GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT GTA ACT ATA 692 Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe Val Thr He 180 185 190 195 ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT GGA AAT TTA 740
Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn Gly Asn Leu 200 205 210
ATA GAT AAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT GTT AGT GAC 788
He Asp Lys Lys Ser He Leu Asn Leu Gly Asn He His Val Ser Asp 215 220 225
AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA TAT ATT GGT 836
Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg Tyr He Gly 230 235 240
ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA ACA GAA ATT 884
He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu Thr Glu He
245 250 255
CAA ACT TTA TAT AAC AAT GAA CCT AAT GCA AAT ATT TTA AAG GAT TTT 932
Gin Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn He Leu Lys Asp Phe 260 265 270 275
TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA TTA AAT GTG 980
Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu Asn Val 280 285 290 TTA AAA CCA AAT AAC TTT ATT AAT AGG AGA ACA GAT TCT ACT TTA AGC 1028
Leu Lys Pro Asn Asn Phe He Asn Arg Arg Thr Asp Ser Thr Leu Ser 295 300 305
ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA TTA TAT AGT 1076 He Asn Asn He Arg Ser Thr He Leu Leu Ala Aεn Arg Leu Tyr Ser 310 315 320
GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT ACT AAC GAT 1124
Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser Thr Asn Asp 325 330 335
AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT GTA GCC AGC 1172
Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe Val Ala Ser 340 345 350 355
AAA ACT CAC TTA CTT CCA TTA TAT GCT GAT ACA GCT ACC ACA AAT AAA 1220
Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys 360 365 370 GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT AAT CAA GTA 1268
Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe Asn Gin Val 375 380 385
GTA GTT ATG AAT TCA GTA GGA AAT TGT ACA ATG AAT TTT AAA AAT AAT 1316 Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe Lys Asn Asn 390 395 400
AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT GTA GTT 1364
Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala Asp Thr Val Val 405 410 415
GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT AAT ACA AAC AGC AAT 1412
Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn Ser Asn 420 425 430 435
GGA TTT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA GAA AAA 1460
Gly Phe Phe Trp Asn Phe He Ser Glu Glu His Gly Trp Gin Glu Lys 440 445 450 TAA 1463
( 2 ) INFORMATION FOR SEQ ID NO : 54 :
U ) SEQUENCE CHARACTERISTICS : ( A) LENGTH : 4 51 ammo acids
525 (B) TYPE: amino acid (D) TOPOLOGY: linear
Ui) MOLECULE TYPE: prote
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15
He Glu Gly Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp 20 25 30
Asp Lys He Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lyε 35 40 45
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 50 55 ' 60
Thr Ser Gly Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys 65 70 75 80
Tyr Pro Thr Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lys Leu Ser 85 90 95
Glu Val Asn He Ser Gin Asn Asp Tyr He He Tyr Asp Asn Lys Tyr 100 105 ' 110
Lys Asn Phe Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn 115 120 125
Lvs He Val Aεn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg 130 135 140 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He 145 150 ' 155 160
Trp Thr Leu Gin Asp Asn Ser Gly He Asn Gin Lys Leu Ala Phe Asn
165 170 175
Tyr Gly Asn Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe
180 185 190
Val Thr He Thr Asn Asp Arg Leu Gly Asp Ser Lyε Leu Tyr He Asn L95 200 205
Gly Asn Leu He Asp Lys Lys Ser He Leu Asn Leu GJ y Asn He His
210 215 220 Val Ser Asp Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg
225 230 235 240
Tyr He Gly He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu 245 250 255
Thr Glu He Gin Thr Leu Tyr Asn Asn Glu Pro Asn Ala Asn He Leu 260 265 270
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu " 275 280 285
Leu Asn Val Leu Lyε Pro Asn Asn Phe He Asn Arg Arg Thr Asp Ser 290 295 300 Thr Leu Ser He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg 305 310 315 320
Leu Tyr Ser Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser 325 330 335 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe
340 345 350
Val Ala Ser Lys Thr His Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr
355 360 365
Thr Asn Lys Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe
370 375 380
Asn Gin Val Val Val Met Aεn Ser Val Gly Asn Cys Thr Met Asn Phe
385 390 395 400
Lys Asn Asn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala Asp 405 410 ' 415
Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr Hiε Met Arg Asp Asn Thr
420 425 430
Asn Ser Asn Gly Phe Phe Trp Asn Phe He Ser Glu Glu His Gly Trp 435 440 445
Gin Glu Lys 450 i?) INFORMATION FOR SEQ ID NO : 55 :
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1472 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
Uκ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 108..1463 (κi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116 Met Gly His
1
CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His He Glu Gly 5 10 15
CGT CAT ATG GCT AGC ATG GCT CTT TCT TCT TAT ACA GAT GAT AAA ATT 212
Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp Asp Lys He 20 25 30 35
TTA ATT TCA TAT TTT AAT AAA TTC TTT AAG AGA ATT AAA AGT AGT TCA 260 Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lys Ser Ser Ser 40 45 50 GTT TTA AAT ATG AGA TAT AAA AAT GAT AAA TAC GTA GAT ACT TCA GGA 308 Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Glv 55 60 65
TAT GAT TCA AAT ATA AAT ATT AAT GGA GAT GTA TAT AAA TAT CCA ACT 356 Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Lys Tyr Pro Thr 70 75 80
AAT AAA AAT CAA TTT GGA ATA TAT AAT GAT AAA CTT AGT GAA GTT AAT 404 Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lys Leu Ser Glu Val Asn 85 90 95 ATA TCT CAA AAT GAT TAC ATT ATA TAT GAT AAT AAA TAT AAA AAT TTT 452 He Ser Gin Asn Asp Tyr He He Tyr Asp Asn Lys Tyr Lys Asn Phe 100 105 110 * 115 AGT ATT AGT TTT TGG GTA AGA ATT CCT AAC TAT GAT AAT AAG ATA GTA 500 Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn Lys He Val 120 125 ' 130
AAT GTT AAT AAT GAA TAC ACT ATA ATA AAT TGT ATG AGA GAT AAT AAT 548 Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg Asp Aεn Asn
135 140 145
TCA GGA TGG AAA GTA TCT CTT AAT CAT AAT GAA ATA ATT TGG ACA TTG 596 Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He Trp Thr Leu 150 155 160
CAA GAT AAT GCA GGA ATT AAT CAA AAA TTA GCA TTT AAC TAT GGT AAC 644
Gin Asp Asn Ala Gly He Asn Gin Lys Leu Ala Phe Asn Tyr Gly Asn
165 170 175
GCA AAT GGT ATT TCT GAT TAT ATA AAT AAG TGG ATT TTT GTA ACT ATA 692
Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe Val Thr He 180 185 190 195 ACT AAT GAT AGA TTA GGA GAT TCT AAA CTT TAT ATT AAT GGA AAT TTA 740 Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn Gly Asn Leu 200 205 210
ATA GAT CAA AAA TCA ATT TTA AAT TTA GGT AAT ATT CAT GTT AGT GAC 788 He Asp Gin Lys Ser He Leu Asn Leu Gly Asn He His Val Ser Asp
215 220 225
AAT ATA TTA TTT AAA ATA GTT AAT TGT AGT TAT ACA AGA TAT ATT GGT 836 Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg Tyr He Gly 230 235 240 '
ATT AGA TAT TTT AAT ATT TTT GAT AAA GAA TTA GAT GAA ACA GAA ATT 884
He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu Thr Glu He
245 250 255
CAA ACT TTA TAT AGC AAT GAA CCT AAT ACA AAT ATT TTG AAG GAT TTT 932
Gin Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn He Leu Lys Aεp Phe
260 265 270 275 TGG GGA AAT TAT TTG CTT TAT GAC AAA GAA TAC TAT TTA TTA AAT GTG 980 Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu Leu Asn Val 280 ' 285 290
TTA AAA CCA AAT AAC TTT ATT GAT AGG AGA AAA GAT TCT ACT TTA AGC 1028 Leu Lys Pro Asn Asn Phe He Asp Arg Arg Lys Asp Ser Thr Leu Ser
295 300 305
ATT AAT AAT ATA AGA AGC ACT ATT CTT TTA GCT AAT AGA TTA TAT AGT 1076 He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg Leu Tyr Ser 310 315 320
GGA ATA AAA GTT AAA ATA CAA AGA GTT AAT AAT AGT AGT ACT AAC GAT 1124
Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser Thr Asn Asp 325 330 335
AAT CTT GTT AGA AAG AAT GAT CAG GTA TAT ATT AAT TTT GTA GCC AGC 1172
Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe Val Ala Ser 340 345 350 355 AAA ACT CAC TTA TTT CCA TTA TAT GCT GAT ACA GCT ACC ACA AAT AAA 1220 Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys 360 * 365 370
GAG AAA ACA ATA AAA ATA TCA TCA TCT GGC AAT AGA TTT AAT CAA GTA 1268 Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe Asn Gin Val 3 75 380 385
GTA GTT ATG AAT TCA GTA GGA AAT AAT TGT ACA ATG AAT TTT AAA AAT 1316 Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn 390 395 400
AAT AAT GGA AAT AAT ATT GGG TTG TTA GGT TTC AAG GCA GAT ACT GTA 1364 Asn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala Asp Thr Val 405 410 415
GTT GCT AGT ACT TGG TAT TAT ACA CAT ATG AGA GAT CAT ACA AAC AGC 1412 Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr Asn Ser 420 425 430 435
AAT GGA TGT TTT TGG AAC TTT ATT TCT GAA GAA CAT GGA TGG CAA GAA 1460 Asn Gly Cys Phe Trp Asn Phe He Ser Glu Glu His Gly Trp Gin Glu 440 445 450
AAA TAAAAGCTT 1472
Lys
( 2 ) INFORMATION FOR SEQ ID NO: 56:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 452 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(l.) MOLECULE TYPE: protem
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Gly His His His His His His His His His His Ser Ser Gly His I S 10 15
He Glu Gly Arg His Met Ala Ser Met Ala Leu Ser Ser Tyr Thr Asp 20 25 30
Asp Lys He Leu He Ser Tyr Phe Asn Lys Phe Phe Lys Arg He Lys 35 40 45
Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp
50 55 60 nr Ser Gly Tyr Asp Ser Asn He Asn He Asn Gly Asp Val Tyr Ly
65 70 75
Tyr Pro Thr Asn Lys Asn Gin Phe Gly He Tyr Asn Asp Lyε Leu Ser
85 90 95
Glu Val Asn He Ser Gin Aεn Asp Tyr He He Tyr Asp Asn Lys Tyr
100 105 110
Lys Asn Phe Ser He Ser Phe Trp Val Arg He Pro Asn Tyr Asp Asn 115 120 125
Lys He Val Asn Val Asn Asn Glu Tyr Thr He He Asn Cys Met Arg 130 135 140 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu He He 145 150 155 160
Trp Thr Leu Gin Asp Asn Ala Gly He Asn Gin Lys Leu Ala Phe Asn
165 170 ' 175
Tyr Gly Asn Ala Asn Gly He Ser Asp Tyr He Asn Lys Trp He Phe 180 185 190
Val Thr He Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr He Asn 195 200 205 Gly Asn Leu He Asp Gin Lys Ser He Leu Asn Leu Gly Asn He His 210 215 220
Val Ser Asp Asn He Leu Phe Lys He Val Asn Cys Ser Tyr Thr Arg 225 230 235 240
Tyr He Gly He Arg Tyr Phe Asn He Phe Asp Lys Glu Leu Asp Glu 245 250 255
Thr Glu He Gin Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn He Leu 260 265 270
Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu 275 280 285
Leu Asn Val Leu Lys Pro Asn Asn Phe He Asp Arg Arg Lys Asp Ser 290 295 300
Thr Leu Ser He Asn Asn He Arg Ser Thr He Leu Leu Ala Asn Arg 305 310 315 320
Leu Tyr Ser Gly He Lys Val Lys He Gin Arg Val Asn Asn Ser Ser 325 330 335
Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gin Val Tyr He Asn Phe 340 345 350
Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr 355 360 365
Thr Asn Lys Glu Lys Thr He Lys He Ser Ser Ser Gly Asn Arg Phe 370 375 380
Asn Gin Val Val Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn 385 390 395 400
Phe Lys Asn Asn Asn Gly Asn Asn He Gly Leu Leu Gly Phe Lys Ala 405 410 415 Asp Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His
420 425 430
Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe He Ser Glu Glu His Gly 435 440 445
Trp Gin Glu Lyε 450
(2) INFORMATION FOR SEQ ID NO : 57
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 57-
CGCCATGGCT CTTTCTTCTT ATACAGATGA T 31
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
GCAAGCTTTT ATTTTTCTTG CCATCCATG 29
(2) INFORMATION FOR SEQ ID NO : 59 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 3876 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: DNA (genomic)
(IX) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 1..3873
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
ATG CCA ATA ACA ATT AAC AAC TTT AAT TAT TCA GAT CCT GTT GAT AAT 48 Met Pio He Thr He Asn Asn Phe Asn Tyr Ser Asp Pro Val Aεp Asn 1 5 10 15
AAA AAT ATT TTA TAT TTA GAT ACT CAT TTA AAT ACA CTA GCT AAT GAG 96
Lys Aεn He Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30
CCT GAA AAA GCC TTT CGC ATT ACA GGA AAT ATA TGG GTA ATA CCT GAT 144 Pro Glu Lys Ala Phe Arg He Thr Gly Asn He Trp Val He Pro Asp 35 40 45
AGA TTT TCA AGA AAT TCT AAT CCA AAT TTA AAT AAA CCT CCT CGA GTT 192 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60 ACA AGC CCT AAA AGT GGT TAT TAT GAT CCT AAT TAT TTG AGT ACT GAT 240 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80
TCT GAC AAA GAT ACA TTT TTA AAA GAA ATT ATA AAG TTA TTT AAA AGA 288 Ser Asp Lys Asp Thr Phe Leu Lys Glu He He Lys Leu Phe Lys Arg
85 90 95
ATT AAT TCT AGA GAA ATA GGA GAA GAA TTA ATA TAT AGA CTT TCG ACA 336 He Aεn Ser Arg Glu He Gly Glu Glu Leu He Tyr Arg Leu Ser Thr 100 105 110
GAT ATA CCC TTT CCT GGG AAT AAC AAT ACT CCA ATT AAT ACT TTT GAT 384
Asp He Pro Phe Pro Gly Asn Asn Asn Thr Pro He Asn Thr Phe Asp 115 120 125
TTT GAT GTA GAT TTT AAC AGT GTT GAT GTT AAA ACT AGA CAA GGT AAC 432
Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gin Gly Asn 130 135 140 AAC TGG GTT AAA ACT GGT AGC ATA AAT CCT AGT GTT ATA ATA ACT GGA 480 Asn Trp Val Lys Thr Gly Ser He Asn Pro Ser Val He He Thr Gly 145 150 155 160
CCT AGA GAA AAC ATT ATA GAT CCA GAA ACT TCT ACG TTT /AA TTA ACT 528 Pro Arg Glu Asn He He Asp Pro Glu Thr Ser Thr Phe Lyε Leu Thr
165 170 175
J_ | AAC AAT ACT TTT GCG GCA CAA GAA GGA TTT GGT GCT TTA TCA ATA ATT 576 Asn Asn Thr Phe Ala Ala Gin Glu Gly Phe Gly Ala Leu Ser He He 180 185 190
TCA ATA TCA CCT AGA TTT ATG CTA ACA TAT AGT AAT GCA ACT AAT GAT 624 Ser He Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205
GTA GGA GAG GGT AGA TTT TCT AAG TCT GAA TTT TGC ATG GAT CCA ATA 672 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro He 210 215 220
CTA ATT TTA ATG CAT GAA CTT AAT CAT GCA ATG CAT AAT TTA TAT GGA 720 Leu He Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 240
ATA GCT ATA CCA AAT GAT CAA ACA ATT TCA TCT GTA ACT AGT AAT ATT 768 He Ala He Pro Asn Asp Gin Thr He Ser Ser Val Thr Ser Asn He 245 250 255
TTT TAT TCT CAA TAT AAT GTG AAA TTA GAG TAT GCA GAA ATA TAT GCA 816 Phe Tyr Ser Gin Tyr Asn Val Lys Leu Glu Tyr Ala Glu He Tyr Ala 260 265 270 TTT GGA GGT CCA ACT ATA GAC CTT ATT CCT AAA AGT GCA AGG AAA TAT 864 Plie Gly Gly Pro Thr He Asp Leu He Pro Lys Ser Ala Arg Lys Tyr 275 280 285
TTT GAG GAA AAG GCA TTG GAT TAT TAT AGA TCT ATA GCT AAA AGA CTT 912 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser He Ala Lys Arg Leu 290 295 300
AAT AGT ATA ACT ACT GCA AAT CCT TCA AGC TTT AAT AAA TAT ATA GGG 960 Asn Ser He Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr He Gly 305 310 315 320
GAA TAT AAA CAG AAA CTT ATT AGA AAG TAT AGA TTC GTA GTA GAA TCT 1008
Glu Tyr Lys Gin Lys Leu He Arg Lys Tyr Arg Phe Val Val Glu Ser
325 330 335
TCA GGT GAA GTT ACA GTA AAT CGT AAT AAG TTT GTT GAG TTA TAT AAT 1056
Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn
340 345 350 GAA CTT ACA CAA ATA TTT ACA GAA TTT AAC TAC GCT AAA ATA TAT AAT 1104 Glu Leu Thr Gin He Phe Thr Glu Phe Asn Tyr Ala Lys He Tyr Asn 355 360 365
GTA CAA AAT AGG AAA ATA TAT CTT TCA AAT GTA TAT ACT CCG GTT ACG 1152 Val Gin Asn Arg Lyε He Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380
GCG AAT ATA TTA GAC GAT AAT GTT TAT GAT ATA CAA AAT GGA TTT AAT 1200 Ala Asn He Leu Asp Asp Asn Val Tyr Asp He Gin Asn Gly Phe Asn 385 390 395 400
ATA CCT AAA AGT AAT TTA AAT GTA CTA TTT ATG GGT CAA AAT TTA TCT 1248 He Pro Lys Ser Asn Leu Aεn Val Leu Phe Met Gly Gin Asn Leu Ser 405 410 415
CGA AAT CCA GCA TTA AGA AAA GTC AAT CCT GAA AAT ATG CTT TAT TTA 1296 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430 TTT ACA AAA TTT TGT CAT AAA GCA ATA GAT GGT AGA TCA TTA TAT AAT 1344 Phe Thr Lys Phe Cys His Lys Ala He Asp Gly Arg Ser Leu Tyr Asn 435 440 445
AAA ACA TTA GAT TGT AGA GAG CTT TTA GTT AAA AAT ACT GAC TTA CCC 1392 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460
TTT ATA GGT GAT ATT AGT GAT GTT AAA ACT GAT ATA TTT TTA AGA AAA 1440
Phe He Gly Asp He Ser Asp Val Lys Thr Asp He Phe Leu Arg Lys
465 470 475 480
GAT ATT AAT GAA GAA ACT GAA GTT ATA TAC TAT CCG GAC AAT GTT TCA 1488
Asp He Asn Glu Glu Thr Glu Val He Tyr Tyr Pro Asp Asn Val Ser 485 490 495
GTA GAT CAA GTT ATT CTC AGT AAG AAT ACC TCA GAA CAT GGA CAA CTA 1536
Val Asp Gin Val He Leu Ser Lys Asn Thr Ser Glu His Gly Gin Leu 500 505 510 GAT TTA TTA TAC CCT AGT ATT GAC AGT GAG AGT GAA ATA TTA CCA GGG 1584
Asp Leu Leu Tyr Pro Ser He Asp Ser Glu Ser Glu He Leu Pro Gly 515 520 525
GAG AAT CAA GTC TTT TAT GAT AAT AGA ACT CAA AAT GTT GAT TAT TTG 1632 Glu Asn Gin Val Phe Tyr Asp Asn Arg Thr Gin Asn Val Asp Tyr Leu 530 535 540
AAT TCT TAT TAT TAC CTA GAA TCT CAA AAA CTA AGT GAT AAT GTT GAA 1680
Asn Ser Tyr Tyr Tyr Leu Glu Ser Gin Lys Leu Ser Asp Asn Val Glu 545 ' 550 555 560
GAT TTT ACT TTT ACG AGA TCA ATT GAG GAG GCT TTG GAT AAT AGT GCA 1728
Asp Phe Thr Phe Thr Arg Ser He Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575
AAA GTA TAT ACT TAC TTT CCT ACA CTA GCT AAT AAA GTA AAT GCG GGT 1776
Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 ' 585 590 GTT CAA GGT GGT TTA TTT TTA ATG TGG GCA AAT GAT GTA GTT GAA GAT 1824
Val Gin Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 ' 600 605
TTT ACT ACA AAT ATT CTA AGA AAA GAT ACA TTA GAT AAA ATA TCA GAT 1872 Phe Thr Thr Asn He Leu Arg Lys Asp Thr Leu Asp Lys He Ser Asp 610 615 620
GTA TCA GCT ATT ATT CCC TAT ATA GGA CCC GCA TTA AAT ATA AGT AAT 1920
Val Ser Ala He He Pro Tyr He Gly Pro Ala Leu Asn He Ser Asn 625 630 635 640
TCT GTA AGA AGA GGA AAT TTT ACT GAA GCA TTT GCA GTT ACT GGT GTA 1968
Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655
ACT ATT TTA TTA GAA GCA TTT CCT GAA TTT ACA ATA CCT GCA CTT GGT 2016
Thr He Leu Leu Glu Ala Phe Pro Glu Phe Thr He Pro Ala Leu Gly 660 665 670 GCA TTT GTG ATT TAT AGT AAG GTT CAA GAA AGA AAC GAG ATT ATT AAA 2064
Ala Phe Val He Tyr Ser Lys Val Gin Glu Arg Asn Glu He He Lys 675 680 685
ACT ATA GAT AAT TGT TTA GAA CAA AGG ATT AAG AGA TGG AAA GAT TCA 2112 Thr He Asp Asn Cys Leu Glu Gin Arg He Lys Arg Trp Lys Asp Ser 690 ■ 695 ' 700
TAT GAA TGG ATG ATG GGA ACG TGG TTA TCC AGG ATT ATT ACT CAA TTT 2160
Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg He He Thr Gin Phe 705 710 715 720
AAT AAT ATA AGT TAT CAA ATG TAT GAT TCT TTA AAT TAT CAG GCA GGT 2208
Asn Asn He Ser Tyr Gin Met Tyr Asp Ser Leu Asn Tyr Gin Ala Gly 725 730 735 GCA ATC AAA GCT AAA ATA GAT TTA GAA TAT AAA AAA TAT TCA GGA AGT 2256 Ala He Lys Ala Lys He Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 GAT AAA GAA AAT ATA AAA AGT CAA GTT GAA AAT TTA AAA AAT AGT TTA 2304 Asp Lys Glu Asn He Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu 755 760 765
GAT GTA AAA ATT TCG GAA GCA ATG AAT AAT ATA AAT AAA TTT ATA CGA 2352 Asp Val Lys He Ser Glu Ala Met Asn Asn He Aεn Lys Phe He Arg 770 775 780
GAA TGT TCC GTA ACA TAT TTA TTT AAA AAT ATG TTA CCT AAA GTA ATT 2400 Glu Cys Ser Val Thr Tyr Leu Phe Lyε Asn Met Leu Pro Lys Val He 785 790 795 ' 800
GAT GAA TTA AAT GAG TTT GAT CGA AAT ACT AAA GCA AAA TTA ATT AAT 2448 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu He Asn 805 810 815
CTT ATA GAT AGT CAT AAT ATT ATT CTA GTT GGT GAA GTA GAT AAA TTA 2496 Leu He Asp Ser His Asn He He Leu Val Gly Glu Val Asp Lys Leu 820 825 830 AAA GCA AAA GTA AAT AAT AGC TTT CAA AAT ACA ATA CCC TTT AAT ATT 2544 Lys Ala Lys Val Asn Asn Ser Phe Gin Asn Thr He Pro Phe Asn He 835 840 845
TTT TCA TAT ACT AAT AAT TCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 2592 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp He He Asn Glu Tyr 850 855 860
TTC AAT AAT ATT AAT GAT TCA AAA ATT TTG AGC CTA CAA AAC AGA AAA 2640 Phe Asn Asn He Asn Asp Ser Lys He Leu Ser Leu Gin Asn Arg Lys 865 870 875 880
AAT ACT TTA GTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGT GAA GAA 2688 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895
GGC GAT GTT CAG CTT AAT CCA ATA TTT CCA TTT GAC TTT AAA TTA GGT 2736 Gly Asp Val Gin Leu Asn Pro He Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910 AGT TCA GGG GAG GAT AGA GGT AAA GTT ATA GTA ACC CAG AAT GAA AAT 2784 Ser Ser Gly Glu Asp Arg Gly Lys Val He Val Thr Gin Asn Glu Asn 915 920 925
ATT GTA TAT AAT TCT ATG TAT GAA AGT TTT AGC ATT AGT TTT TGG ATT 2832 He Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser He Ser Phe Trp He 930 935 940
AGA ATA AAT AAA TGG GTA AGT AAT TTA CCT GGA TAT ACT ATA ATT GAT 2880 Arg He Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr He He Asp 945 950 955 960
AGT GTT AAA AAT AAC TCA GGT TGG AGT ATA GGT ATT ATT AGT AAT TTT 2928
Ser Val Lys Asn Asn Ser Gly Trp Ser He Gly He He Ser Asn Phe
965 970 975
TTA GTA TTT ACT TTA AAA CAA AAT GAA GAT AGT GAA CAA AGT ATA AAT 2976
Leu Val Phe Thr Leu Lys Gin Asn Glu Asp Ser Glu Gin Ser He Asn 980 985 990 TTT AGT TAT GAT ATA TCA AAT AAT GCT CCT GGA TAC AAT AAA TGG TTT 3024 Phe Ser Tyr Asp He Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005
TTT GTA ACT GTT ACT AAC AAT ATG ATG GGA AAT ATG AAG ATT TAT ATA 3072 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys He Tyr He
- J 34 - 1010 1015 1020
AAT GGA AAA TTA ATA GAT ACT ATA AAA GTT AAA GAA CTA ACT GGA ATT 3120 Asn Gly Lys Leu He Asp Thr He Lys Val Lys Glu Leu Thr Gly He 1025 1030 1035 1040
AAT TTT AGC AAA ACT ATA ACA TTT GAA ATA AAT AAA ATT CCA GAT ACC 3168 Asn Phe Ser Lys Thr He Thr Phe Glu He Asn Lys He Pro Asp Thr 1045 1050 1055
GGT TTG ATT ACT TCA GAT TCT GAT AAC ATC AAT ATG TGG ATA AGA GAT 3216 Gly Leu He Thr Ser Asp Ser Asp Asn He Asn Met Trp He Arg Asp 1060 1065 1070 TTT TAT ATA TTT GCT AAA GAA TTA GAT GGT AAA GAT ATT AAT ATA TTA 3264 Phe Tyr He Phe Ala Lys Glu Leu Asp Gly Lys Asp He Asn He Leu 1075 1080 1085
TTT AAT AGC TTG CAA TAT ACT AAT GTT GTA AAA GAT TAT TGG GGA AAT 3312 Phe Asn Ser Leu Gin Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090 1095 1100
GAT TTA AGA TAT AAT AAA GAA TAT TAT ATG GTT AAT ATA GAT TAT TTA 3360 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn He Asp Tyr Leu 1105 1110 1115 1120
AAT AGA TAT ATG TAT GCG AAC TCA CGA CAA ATT GTT TTT AAT ACA CGT 3408 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gin He Val Phe Aεn Thr Arg 1125 1130 1135
AGA AAT AAT AAT GAC TTC AAT GAA GGA TAT AAA ATT ATA ATA AAA AGA 3456 Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys He He He Lys Arg 1140 1145 1150 ATC AGA GGA AAT ACA AAT GAT ACT AGA GTA CGA GGA GGA GAT ATT TTA 3504 He Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp He Leu 1155 1160 1165
TAT TTT GAT ATG ACA ATT AAT AAC AAA GCA TAT AAT TTG TTT ATG AAG 3552 Tyr Phe Asp Met Thr He Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys 1170 1175 1180
AAT GAA ACT ATG TAT GCA GAT AAT CAT AGT ACT GAA GAT ATA TAT GCT 3600 Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp He Tyr Ala 1185 1190 1195 1200
ATA GGT TTA AGA GAA CAA ACA AAG GAT ATA AAT GAT AAT ATT ATA TTT 3648
He Gly Leu Arg Glu Gin Thr Lys Asp He Asn Asp Asn He He Phe
1205 1210 1215
CAA ATA CAA CCA ATG AAT AAT ACT TAT TAT TAC GCA TCT CAA ATA TTT 3696
Gin He Gin Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gin He Phe
1220 1225 1230 AAA TCA AAT TTT AAT GGA GAA AAT ATT TCT GGA ATA TGT TCA ATA GGT 3744 Lys Ser Asn Phe Asn Gly Glu Asn He Ser Gly He Cys Ser He Gly 1235 1240 1245
ACT TAT CGT TTT AGA CTT GGA GGT GAT TGG TAT AGA CAC AAT TAT TTG 3792 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250 • 1255 1260
GTG CCT ACT GTG AAG CAA GGA AAT TAT GCT TCA TTA TTA GAA TCA ACA 3840 Val Pro Thr Val Lys Gin Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 1265 1270 1275 1280
TCA ACT CAT TGG GGT TTT GTA CCT GTA AGT GAA TAA 3876
Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1285 1290 (2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1291 am o acids (B) TYPE: ammo acid
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: protein (χι) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Met Pro He Thr He Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn
1 5 10 15 Lys Asn He Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu
20 25 30
Pro Glu Lys Ala Phe Arg He Thr Gly Asn He Trp Val He Pro Asp
35 40 45
Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val
50 55 60
Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp
65 70 75 80
Ser Asp Lys Asp Thr Phe Leu Lys Glu He He Lys Leu Phe Lys Arg 85 90 95
He Asn Ser Arg Glu He Gly Glu Glu Leu He Tyr Arg Leu Ser Thr 100 105 110
Asp He Pro Phe Pro Gly Asn Asn Asn Thr Pro He Asn Thr Phe Asp 115 120 125
Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gin Gly Asn 130 135 140
Asn Trp Val Lys Thr Gly Ser He Asn Pro Ser Val He He Thr Gly 145 150 155 160
Pro Arg Glu Asn He He Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr
165 170 175 Asn Asn Thr Phe Ala Ala Gin Glu Gly Phe Gly Ala Leu Ser He He
180 185 190
Ser He Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp
195 200 205
Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro He 210 215 220
Leu He Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 ' 240
He Ala He Pro Asn Asp Gin Thr He Ser Ser Val Thr Ser Asn He 245 250 255 Phe Tvr Ser Gin Tyr Asn Val Lys Leu Glu Tyr Ala Glu He Tyr Ala
260 265 270
Phe Gly Gly Pro Thr He Asp Leu He Pro Lys Ser Ala Arg Lys Tyr
275 280 285
Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser He Ala Lys Arg Leu
290 295 300
Asn Ser He Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr He Gly 305 310 315 320 Glu Tyr Lys Gin Lys Leu He Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335
Ser Gly Glu Val Thr Val Asn Arg Asn Lyε Phe Val Glu Leu Tyr Asn 340 345 350
Glu Leu Thr Gin He Phe Thr Glu Phe Asn Tyr Ala Lys He Tyr Asn 355 360 365
Val Gin Asn Arg Lys He Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380
Ala Asn He Leu Asp Asp Asn Val Tyr Asp He Gin Asn Gly Phe Asn 385 390 395 400
He Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gin Asn Leu Ser 405 410 415
Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430
Phe Thr Lys Phe Cys His Lys Ala He Asp Gly Arg Ser Leu Tyr Asn 435 440 445
Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Aεn Thr Aεp Leu Pro 450 455 460 he He Gly Aεp He Ser Asp Val Lys Thr Asp He Phe Leu Arg Lys 465 470 475 480
Asp He Asn Glu Glu Thr Glu Val He Tyr Tyr Pro Asp Asn Val Ser 485 490 495
Val Asp Gin Val He Leu Ser Lys Asn Thr Ser Glu His Gly Gin Leu 500 505 510
Asp Leu Leu Tyr Pro Ser He Asp Ser Glu Ser Glu He Leu Pro Gly 515 520 525 Glu Asn Gin Val Phe Tyr Asp Asn Arg Thr Gin Asn Val Asp Tyr Leu 530 ' 535 540
Asn Ser Tyr Tyr Tyr Leu Glu Ser Gin Lys Leu Ser Asp Asn Val Glu 545 550 555 560
3p Phe Thr Phe Thr Arg Ser He Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575
Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Aεn Ala Gly 580 585 590
Val Gin Gly Gly Leu Phe Leu Met Trp Ala Asn Aεp Val Val Glu Asp
595 600 * 605 Phe Thr Thr Asn He Leu Arg Lys Asp Thr Leu Asp Lys He Ser Asp
610 615 620
Val Ser Ala He He Pro Tyr He Gly Pro Ala Leu Asn He Ser Asn
625 630 635 640
Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val
645 650 655
Thr He Leu Leu Glu Ala Phe Pro Glu Phe Thr He Pro Ala Leu Gly 660 665 670
Ala Phe Val He Tyr Ser Lys Val Gin Glu Arg Asn Glu He He Lys
675 680 685 Thr He Asp Asn Cys Leu Glu Gin Arg He Lys Arg Trp Lys Asp Ser
- .15 / - 6 90 6 95 700
Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg He He Thr Gin Phe 705 710 715 720
Asn Asn He Ser Tyr Gin Met Tyr Asp Ser Leu Asn Tyr Gin Ala Gly 725 730 735
Ala He Lys Ala Lys He Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750
Asp Lys Glu Asn He Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 Asp Val Lys He Ser Glu Ala Met Asn Asn He Asn Lys Phe He Arg 770 775 780
Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val He 785 790 795 800
Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu He Asn 805 810 815
Leu He Asp Ser His Asn He He Leu Val Gly Glu Val Asp Lys Leu 820 825 830
Lys Ala Lys Val Asn Asn Ser Phe Gin Asn Thr He Pro Phe Asn He 835 840 845 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp He He Asn Glu Tyr 850 855 860
Phe Asn Asn He Asn Asp Ser Lys He Leu Ser Leu Gin Asn Arg Lys 865 870 875 880
Asn Thr Leu Val Aεp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895
Gly Asp Val Gin Leu Asn Pro He Phe Pro Phe Asp Phe Lyε Leu Gly 900 905 910
Ser Ser Gly Glu Asp Arg Gly Lys Val He Val Thr Gin Asn Glu Asn 915 920 925 He Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser He Ser Phe Trp He 930 935 940
Arg He Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr He He Asp 945 950 955 960
Ser Val Lys Asn Asn Ser Gly Trp Ser He Gly He He Ser Asn Phe 965 970 975
Leu Val Phe Thr Leu Lys Gin Asn Glu Asp Ser Glu Gin Ser He Asn 980 985 990
Phe Ser Tyr Asp He Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys He Tyr He 1010 1015 1020
Asn Gly Lys Leu He Asp Thr He Lys Val Lys Glu Leu Thr Gly He 1025 1030 1035 1040
Asn Phe Ser Lys Thr He Thr Phe Glu He Asn Lys He Pro Asp Thr 1045 1050 1055
Gly Leu He Thr Ser Asp Ser Asp Asn He Asn Met Trp He Arg Asp 1060 1065 1070 Phe Tyr He Phe Ala Lys Glu Leu Asp Gly Lys Asp He Asn He Leu 1075 1080 1085
Phe Asn Ser Leu Gin Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 1090 1095 1100
Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn He Asp Tyr Leu 1105 1110 1115 1120 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gin He Val Phe Asn Thr Arg
1125 1130 1135
Arg Asn Asn Asn Aεp Phe Asn Glu Gly Tyr Lys He He He Lys Arg 1140 1145 1150
He Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp He Leu 1155 1160 1165
Tyr Phe Asp Met Thr He Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys ' 1170 1175 1180
Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp He Tyr Ala 1185 1190 1195 1200 He Gly Leu Arg Glu Gin Thr Lys Aεp He Asn Asp Asn He He Phe
1205 1210 1215
Gin He Gin Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gin He Phe 1220 1225 1230
Lys Ser Asn Phe Asn Gly Glu Asn He Ser Gly He Cys Ser He Gly 1235 1240 1245
Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu 1250 1255 1260
Val Pro Thr Val Lys Gin Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 1265 1270 1275 1280 Ser Thr Hiε Trp Gly Phe Val Pro Val Ser Glu
1285 1290
(2) INFORMATION FOR SEQ ID NO: 61: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1502 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 108..1493
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 61 :
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Met Gly His 1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His He Glu Gly 5 10 15
CGT CAT ATG GCT AGC ATG GCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 212 Arg His Met Ala Ser Met Ala Leu Leu Lys Asp He He Asn Glu Tyr 20 2 5 30 35
TTC AAT AAT ATT AAT GAT TCA AAA ATT TTG AGC CTA CAA AAC AGA AAA 260 Phe Asn Asn He Asn Asp Ser Lys He Leu Ser Leu Gin Asn Arg Lys 40 45 50
AAT ACT TTA GTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGT GAA GAA 308 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 55 ' 60 65
GGC GAT GTT CAG CTT AAT CCA ATA TTT CCA TTT GAC TTT AAA TTA GGT 356 Gly Asp Val Gin Leu Asn Pro He Phe Pro Phe Asp Phe Lys Leu Gly 70 75 80 AGT TCA GGG GAG GAT AGA GGT AAA GTT ATA GTA ACC CAG AAT GAA AAT 404 Ser Ser Gly Glu Asp Arg Gly Lys Val He Val Thr Gin Asn Glu Asn 85 90 95
ATT GTA TAT AAT TCT ATG TAT GAA AGT TTT AGC ATT AGT TTT TGG ATT 452 He Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser He Ser Phe Trp He 100 105 110 115
AGA ATA AAT AAA TGG GTA AGT AAT TTA CCT GGA TAT ACT ATA ATT GAT 500 Arg He Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr He He Asp 120 125 130
AGT GTT AAA AAT AAC TCA GGT TGG AGT ATA GGT ATT ATT AGT AAT TTT 548 Ser Val Lys Asn Asn Ser Gly Trp Ser He Gly He He Ser Asn Phe 135 140 145
TTA GTA TTT ACT TTA AAA CAA AAT GAA GAT AGT GAA CAA AGT ATA AAT 596 Leu Val Phe Thr Leu Lys Gin Asn Glu Asp Ser Glu Gin Ser He Asn 150 155 160 TTT AGT TAT GAT ATA TCA AAT AAT GCT CCT GGA TAC AAT AAA TGG TTT 644 Phe Ser Tyr Asp He Ser Asn Aεn Ala Pro Gly Tyr Asn Lyε Trp Phe 165 170 175
TTT GTA ACT GTT ACT AAC AAT ATG ATG GGA AAT ATG AAG ATT TAT ATA 692 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys He Tyr He 180 185 190 195
AAT GGA AAA TTA ATA GAT ACT ATA AAA GTT AAA GAA CTA ACT GGA ATT 740 Asn Gly Lys Leu He Asp Thr He Lys Val Lys Glu Leu Thr Gly He 200 205 210
AAT TTT AGC AAA ACT ATA ACA TTT GAA ATA AAT AAA ATT CCA GAT ACC 788 Asn Phe Ser Lys Thr He Thr Phe Glu He Asn Lys He Pro Asp Thr 215 220 225
GGT TTG ATT ACT TCA GAT TCT GAT AAC ATC AAT ATG TGG ATA AGA GAT 836 Gly Leu He Thr Ser Asp Ser Asp Asn He Asn Met Trp He Arg Asp 230 235 240 TTT TAT ATA TTT GCT AAA GAA TTA GAT GGT AAA GAT ATT AAT ATA TTA 884 Phe Tyr He Phe Ala Lys Glu Leu Asp Gly Lys Asp He Asn He Leu 245 250 ' 255
TTT AAT AGC TTG CAA TAT ACT AAT GTT GTA AAA GAT TAT TGG GGA AAT 932 Phe Asn Ser Leu Gin Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn 260 265 270 275
GAT TTA AGA TAT AAT AAA GAA TAT TAT ATG GTT AAT ATA GAT TAT TTA 980 Asp Leu Arg Tyr Aεn Lys Glu Tyr Tyr Met Val Asn He Asp Tyr Leu 280 285 290
AAT AGA TAT ATG TAT GCG AAC TCA CGA CAA ATT GTT TTT AAT ACA CGT 1028 Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gin He Val Phe Asn Thr Arg 295 300 305 AGA AAT AAT AAT GAC TTC AAT GAA GGA TAT AAA ATT ATA ATA AAA AGA 1076
Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys He He He Lys Arg
310 315 320 ATC AGA GGA AAT ACA AAT GAT ACT AGA GTA CGA GGA GGA GAT ATT TTA 1124
He Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp He Leu
325 330 " 335
TAT TTT GAT ATG ACA ATT AAT AAC AAA GCA TAT AAT TTG TTT ATG AAG 1172 Tyr Phe Asp Met Thr He Asn Asn Lys Ala Tyr Asn Leu Phe Met Lyε
340 345 350 355
AAT GAA ACT ATG TAT GCA GAT AAT CAT AGT ACT GAA GAT ATA TAT GCT 1220
Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp He Tyr Ala 360 365 370
ATA GGT TTA AGA GAA CAA ACA AAG GAT ATA AAT GAT AAT ATT ATA TTT 1268
He Gly Leu Arg Glu Gin Thr Lys Asp He Asn Asp Asn He He Phe
375 ' .380 385
CAA ATA CAA CCA ATG AAT AAT ACT TAT TAT TAC GCA TCT CAA ATA TTT 1 16
Gin He Gin Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gin He Phe
390 395 400 AAA TCA AAT TTT AAT GGA GAA AAT ATT TCT GGA ATA TGT TCA ATA GGT 1364
Lys Ser Asn Phe Asn Gly Glu Asn He Ser Gly He Cys Ser He Gly
405 ' 410 415 "
ACT TAT CGT TTT AGA CTT GGA GGT GAT TGG TAT AGA CAC AAT TAT TTG 1412 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu
420 425 430 435
GTG CCT ACT GTG AAG CAA GGA AAT TAT GCT TCA TTA TTA GAA TCA ACA 1460
Val Pro Thr Val Lys Gin Gly Asn Tyr Ala Ser Leu Leu Glu Ser Thr 440 445 450
"TCA ACT CAT TGG GGT TTT GTA CCT GTA AGT GAA TAAAAGCTT 1502
Ser Thr His Trp Gly Phe Val Pro Val Ser Glu
455 460
INFORMATION FOR SEQ ID NO : 62
' 1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids
Figure imgf000343_0001
(D) TOPOLOGY: linear
(11) MOLECULE TYPE: protem (χι) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 He Glu Gly Arg His Met Ala Ser Met Ala Leu Leu Lys Asp He He
20 25 30
Asn Glu Tyr Phe Asn Asn He Asn Asp Ser Lys He Leu Ser Leu Gin 35 40 45
Asn Arg Lys Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 50 55 60
Ser Glu Glu Gly Asp Val Gin Leu Asn Pro He Phe Pro Phe Asp Phe 65 70 75 80
Lys Leu Gly Ser Ser Gly Glu Asp Arg Gly Lys Val He Val Thr Gin 85 90 95 Asn Glu Asn He Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser He Ser 100 105 110
Phe Trp He Arg He Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr
115 120 125
He He Asp Ser Val Lys Asn Asn Ser Gly Trp Ser He Gly He He 130 135 140
Ser Asn Phe Leu Val Phe Thr Leu Lys Gin Asn Glu Asp Ser Glu Gin 145 150 155 160
Ser He Asn Phe Ser Tyr Asp He Ser Asn Asn Ala Pro Gly Tyr Asn 165 170 175 Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys
180 185 190
He Tyr He Asn Gly Lys Leu He Asp Thr He Lys Val Lys Glu Leu 195 200 205
Thr Gly He Asn Phe Ser Lys Thr He Thr Phe Glu He Asn Lyε He 210 215 220
Pro Aεp Thr Gly Leu He Thr Ser Asp Ser Asp Asn He Aεn Met Trp 225 230 235 240
He Arg Asp Phe Tyr He Phe Ala Lys Glu Leu Asp Gly Lyε Asp l e 245 250 255 Asn He Leu Phe Asn Ser Leu Gin Tyr Thr Asn Val Val Lys Asp Tyr
260 265 270
Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn He 275 280 ' 285
Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gin He Val Phe 290 295 300
Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys He He 305 310 315 320
He Lyε Arg He Aig Gly Asn Thr Asn Asp Thr Arg Val Arg Gly Gly
325 ' 330 335 Asp He Leu Tyr Phe Asp Met Thr He Asn Asn Lys Ala Tyr Asn Leu
340 345 350
Phe Met Lys Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp 355 360 365
He Tyr Ala He Gly Leu Arg Glu Gin Thr Lys Asp He Asn Asp Asn 370 375 380
He He Phe Gin He Gin Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser 385 390 395 400
Gin He Phe Lys Ser Asn Phe Asn Gly Glu Asn He Ser Gly He Cys 405 410 415 Ser He Gly Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His
420 425 430
Asn Tyr Leu Val Pro Thr Val Lys Gin Gly Asn Tyr Ala Ser Leu Leu 435 440 445
Glu Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 450 455 460
(2) INFORMATION FOR SEQ ID NO.63: (l) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 63 :
CGCCATGGCT TTATTAAAAG ATATAATTAA TG
(2) INFORMATION FOR SEQ ID NO: 64:
U) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
GCAAGCTTTT ATTCACTTAC AGGTACAAAA CC 32
12) INFORMATION FOR SEQ ID NO : 65 :
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3831 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS- double (D) TOPOLOGY: linear m) MOLECULE TYPE: DNA (genomic)
Six) FEATURE: (A) NAME /KEY: CDS
(B) LOCATION: 1..3828
(κi) SEQUENCE DESCRIPTION: SEQ ID NO : 65 : ATG ACA TGG CCA GTA AAA GAT TTT AAT TAT AGT GAT CCT GTT AAT GAC 48
Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15
AAT GAT ATA TTA TAT TTA AGA ATA CCA CAA AAT AAG TTA ATT ACT ACA 96 Asn Asp He Leu Tyr Leu Arg He Pro Gin Asn Lys Leu He Thr Thr
20 25 30
CCT GTA AAA GCT TTT ATG ATT ACT CAA AAT ATT TGG GTA ATA CCA GAA 144 Pro Val Lys Ala Phe Met He Thr Gin Asn He Trp Val He Pro Glu 35 40 45
AGA TTT TCA TCA GAT ACT AAT CCA AGT TTA AGT AAA CCG CCC AGA CCT 192 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60
ACT TCA AAG TAT CAA AGT TAT TAT GAT CCT AGT TAT TTA TCT ACT GAT 240 Thr Ser Lys Tyr Gin Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80 GAA CAA AAA GAT ACA TTT TTA AAA GGG ATT ATA AAA TTA TTT AAA AGA 288 Glu Gin Lys Asp Thr Phe Leu Lys Gly He He Lyε Leu Phe Lys Arg 85 90 95
ATT AAT GAA AGA GAT ATA GGA AAA AAA TTA ATA AAT TAT TTA GTA GTT 336 He Aεn Glu Arg Asp He Gly Lys Lyε Leu He Aεn Tyr Leu Val Val - 100 105 110
GGT TCA CCT TTT ATG GGA GAT TCA AGT ACG CCT GAA GAT ACA TTT GAT 384 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125
TTT ACA CGT CAT ACT ACT AAT ATT GCA GTT GAA AAG TTT GAA AAT GGT 432 Phe Thr Arg His Thr Thr Asn He Ala Val Glu Lys Phe Glu Asn Gly 130 135 140
AGT TGG AAA GTA ACA AAT ATT ATA ACA CCA AGT GTA TTG ATA TTT GGA 480 Ser Trp Lys Val Thr Asn He He Thr Pro Ser Val Leu He Phe Gly 145 150 155 160 CCA CTT CCT AAT ATA TTA GAC TAT ACA GCA TCC CTT ACA TTG CAA GGA 528 Pro Leu Pro Asn He Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gin Gly 165 170 175
CAA CAA TCA AAT CCA TCA TTT GAA GGG TTT GGA ACA TTA TCT ATA CTA 576 Gin Gin Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser He Leu
180 185 190
AAA GTA GCA CCT GAA TTT TTG TTA ACA TTT AGT GAT GTA ACA TCT AAT 624 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 * 205
CAA AGT TCA GCT GTA TTA GGC AAA TCT ATA TTT TGT ATG GAT CCA GTA 672 Gin Ser Ser Ala Val Leu Gly Lys Ser He Phe Cys Met Asp Pro Val 210 215 220
ATA GCT TTA ATG CAT GAG TTA ACA CAT TCT TTG CAT CAA TTA TAT GGA 720 He Ala Leu Met His Glu Leu Thr His Ser Leu His Gin Leu Tyr Gly 225 230 235 240 ATA AAT ATA CCA TCT GAT AAA AGG ATT CGT CCA CAA GTT AGC GAG GGA 768 He Asn He Pro Ser Asp Lys Arg He Arg Pro Gin Val Ser Glu Gly 245 250 255
TTT TTC TCT CAA GAT GGA CCC AAC GTA CAA TTT GAG GAA TTA TAT ACA 816 Phe Phe Ser Gin Asp Gly Pro Asn Val Gin Phe Glu Glu Leu Tyr Thr
260 265 270
TTT GGA GGA TTA GAT GTT GAA ATA ATA CCT CAA ATT GAA AGA TCA CAA 864 Phe Gly Gly Leu Asp Val Glu He He Pro Gin He Glu Arg Ser Gin 275 280 285
TTA AGA GAA AAA GCA TTA GGT CAC TAT AAA GAT ATA GCG AAA AGA CTT 912
Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp He Ala Lys Arg Leu 290 295 300
AAT AAT ATT AAT AAA ACT ATT CCT TCT AGT TGG ATT AGT AAT ATA GAT 960
Asn Asn He Asn Lys Thr He Pro Ser Ser Trp He Ser Asn He Asp
305 310 315 320 AAA TAT AAA AAA ATA TTT TCT GAA AAG TAT AAT TTT GAT AAA GAT AAT 1008 Lys Tyr Lys Lys He Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335
ACA GGA AAT TTT GTT GTA AAT ATT GAT AAA TTC AAT AGC TTA TAT TCA 1056 Thr Gly Asn Phe Val Val Asn He Asp Lyε Phe Asn Ser Leu Tyr Sei
340 345 350
544 - GAC TTG ACT AAT GTT ATG TCA GAA GTT GTT TAT TCT TCG CAA TAT AAT 1104
Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gin Tyr Asn 355 360 365 GTT AAA AAC AGG ACT CAT TAT TTT TCA AGG CAT TAT CTA CCT GTA TTT 1152
Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380
GCA AAT ATA TTA GAT GAT AAT ATT TAT ACT ATA AGA GAT GGT TTT AAT 1200 Ala Asn He Leu Asp Asp Asn He Tyr Thr He Arg Asp Gly Phe Asn 385 390 395 400
TTA ACA AAT AAA GGT TTT AAT ATA GAA AAT TCG GGT CAG AAT ATA GAA 1248
Leu Thr Asn Lys Gly Phe Asn He Glu Asn Ser Gly Gin Asn He Glu 405 410 415
AGG AAT CCT GCA CTA CAA AAG CTT AGT TCA GAA AGT GTA GTA GAT TTA 1296
Arg Asn Pro Ala Leu Gin Lys Leu Ser Ser Glu Ser Val Val Asp Leu
420 425 430
TTT ACA AAA GTA TGT TTA AGA TTA ACA AAA AAT AGT AGA GAT GAT TCA 1344
Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445 ACA TGT ATT AAA GTT AAA AAT AAT AGA TTA CCT TAT GTA GCT GAT AAA 1392
Thr Cys He Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460
GAT AGC ATT TCA CAA GAA ATA TTT GAA AAT AAA ATT ATT ACA GAT GAG 1440 Aεp Ser He Ser Gin Glu He Phe Glu Aεn Lys He He Thr Asp Glu 465 470 475 480
ACT AAT GTA CAA AAT TAT TCA GAT AAT TTT TCA TTA GAT GAA TCT ATT 1488
Thr Asn Val Gin Asn Tyr Ser Asp Asn Phe Ser Leu Asp Glu Ser He 485 490 495
TTA GAT GGG CAA GTT CCT ATT AAT CCT GAA ATA GTA GAT CCA CTA TTA 1536
Leu Asp Gly Gin Val Pro He Asn Pro Glu He Val Asp Pro Leu Leu
500 505 510
CCC AAT GTT AAT ATG GAA CCT TTA AAT CTT CCA GGT GAA GAA ATA GTA 1584
Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu He Val 515 520 525 TTT TAT GAT GAT ATT ACT AAA TAT GTT GAT TAT TTA AAT TCT TAT TAT 1632
Phe Tvr Asp Asp He Thr Lyε Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540
TAT TTG GAA TCT CAA AAA TTA AGT AAT AAT GTT GAA AAT ATT ACT CTT 1680 Tyr Leu Glu Ser Gin Lys Leu Ser Asn Asn Val Glu Aεn He Thr Leu 545 550 555 560
ACA ACT TCA GTT GAA GAA GCA TTA GGT TAT AGC AAT AAG ATA TAC ACA 1728
Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys He Tyr Thr 565 570 575
TTT TTA CCT AGC TTA GCT GAA AAA GTG AAT AAA GGT GTT CAA GCA GGT 1776
Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gin Ala Gly
580 585 590
TTA TTC TTA AAT TGG GCG AAT GAA GTA GTT GAG GAT TTT ACT ACA AAT 1824
Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 ATT ATG AAG AAA GAT ACA TTG GAT AAA ATA TCA GAT GTA TCA GTA ATA 1872
He Met Lys Lys Asp Thr Leu Asp Lys He Ser Asp Val Ser Val He 610 615 620
ATT CCA TAT ATA GGA CCT GCC TTA AAT ATA GGA AAT TCA GCA TTA AGG 1920 He Pro Tyr He Gly Pro Ala Leu Asn He Gly Asn Ser Ala Leu Arg 625 630 635 640
GGA AAT TTT AAG CAA GCA TTT GCA ACA GCT GGT GTA GCT TTT TTA TTA 1968 Gly Asn Phe Lys Gin Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655
GAG GGA TTT CCA GAG TTT ACT ATA CCT GCA CTC GGT GTA TTT ACC TTT 2016 Glu Gly Phe Pro Glu Phe Thr He Pro Ala Leu Gly Val Phe Thr Phe 660 665 670
TAT AGT TCT ATT CAA GAA AGA GAG AAA ATT ATT AAA ACT ATA GAA AAT 2064 Tyr Ser Ser He Gin Glu Arg Glu Lys He He Lys Thr He Glu Asn 675 680 685 TGT TTG GAA CAA AGA GTT AAG AGA TGG AAA GAT TCA TAT CAA TGG ATG 2112 Cys Leu Glu Gin Arg Val Lys Arg Trp Lys Asp Ser Tyr Gin Trp Met 690 695 700
GTA TCA AAT TGG TTG TCA AGA ATT ACT ACT CAA TTT AAT CAT ATA AAT 2160 Val Ser Asn Trp Leu Ser Arg He Thr Thr Gin Phe Asn His He Asn 705 710 715 720
TAT CAA ATG TAT GAT TCT TTA AGT TAT CAG GCA GAT GCA ATC AAA GCT 2208 Tyr Gin Met Tyr Asp Ser Leu Ser Tyr Gin Ala Asp Ala He Lys Ala ' 725 730 735
AAA ATA GAT TTA GAA TAT AAA AAA TAC TCA GGA AGT GAT AAA GAA AAT 2256 Lys He Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750
ATA AAA AGT CAA GTT GAA AAT TTA AAA AAT AGT TTA GAT GTA AAA ATT 2304 He Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys He 755 760 765 TCG GAA GCA ATG AAT AAT ATA AAT AAA TTT ATA CGA GAA TGT TCT GTA 2352 Ser Glu Ala Met Asn Asn He Asn Lys Phe He Arg Glu Cys Ser Val 770 775 780
ACA TAC TTA TTT AAA AAT ATG CTC CCT AAA GTA ATT GAC GAA TTA AAT 2400 Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val He Asp Glu Leu Asn 785 ' 790 795 800
AAG TTT GAT TTA AGA ACT AAA ACA GAA TTA ATT AAT CTT ATA GAT AGT 2448 Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu He Asn Leu He Asp Ser 805 810 815
CAT AAT ATT ATT CTA GTT GGT GAA GTA GAT AGA TTA AAA GCA AAA GTA 2496
His Asn He He Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lyε Val
820 825 830
AAT GAG AGT TTT GAA AAT ACA ATG CCT TTT AAT ATT TTT TCA TAT ACT 2544
Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn He Phe Ser Tyr Thr
835 840 845 AAT AAT TCT TTA TTA AAA GAT ATA ATT AAT GAA TAT TTC AAT AGT ATT 2592 Asn Asn Ser Leu Leu Lys Asp He He Asn Glu Tyr Phe Aεn Ser He 850 855 860
AAT GAT TCA AAA ATT TTG AGC TTA CAA AAC AAA AAA AAT GCT TTA GTG 2640 Asn Asp Ser Lys He Leu Ser Leu Gin Asn Lys Lys Asn Ala Leu Val 865 870 875 880
GAT ACA TCA GGA TAT AAT GCA GAA GTG AGG GTA GGA GAT AAT GTT CAA 2688 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gin 885 890 895
CTT AAT ACG ATA TAT ACA AAT GAC TTT AAA TTA AGT AGT TCA GGA GAT 2736
Leu Asn Thr He Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp
900 905 910 AAA ATT ATA GTA AAT TTA AAT AAT AAT ATT TTA TAT AGC GCT ATT TAT 2764 Lys He He Val Asn Leu Asn Asn Asn He Leu Tyr Ser Ala He Tyr 915 920 925
GAG AAC TCT AGT GTT AGT TTT TGG ATT AAG ATA TCT AAA GAT TTA ACT 2832 Glu Asn Ser Ser Val Ser Phe Trp He Lys He Ser Lys Asp Leu Thr 930 935 940
AAT TCT CAT AAT GAA TAT ACA ATA ATT AAC AGT ATA GAA CAA AAT TCT 2880 Asn Ser His Asn Glu Tyr Thr He He Asn Ser He Glu Gin Asn Ser 945 950 955 960
GGG TGG AAA TTA TGT ATT AGG AAT GGC AAT ATA GAA TGG ATT TTA CAA 2928 Gly Trp Lys Leu Cys He Arg Asn Gly Asn He Glu Trp He Leu Gin 965 970 975
GAT GTT AAT AGA AAG TAT AAA AGT TTA ATT TTT GAT TAT AGT GAA TCA 2976 Asp Val Asn Arg Lys Tyr Lys Ser Leu He Phe Asp Tyr Ser Glu Ser 980 985 990
TTA AGT CAT ACA GGA TAT ACA AAT AAA TGG TTT TTT GTT ACT ATA ACT 3024 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr He Thr 995 1000 ' 1005 AAT AAT ATA ATG GGG TAT ATG AAA CTT TAT ATA AAT GGA GAA TTA AAG 3072 Asn Asn He Met Gly Tyr Met Lys Leu Tyr He Asn Gly Glu Leu Lys 1010 1015 1020
CAG AGT CAA AAA ATT GAA GAT TTA GAT GAG GTT AAG TTA GAT AAA ACC 3120 Gin Ser Gin Lys He Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr 1025 1030 1035 1040
ATA GTA TTT GGA ATA GAT GAG AAT ATA GAT GAG AAT CAG ATG CTT TGG 3168 He Val Phe Gly He Asp Glu Asn He Asp Glu Asn Gin Met Leu Trp 1045 1050 1055
ATT AGA GAT TTT AAT ATT TTT TCT AAA GAA TTA AGT AAT GAA GAT ATT 3216
He Arg Asp Phe Asn He Phe Ser Lys Glu Leu Ser Asn Glu Asp He
1060 1065 1070
AAT ATT GTA TAT GAG GGA CAA ATA TTA AGA AAT GTT ATT AAA GAT TAT 3264
Asn He Val Tyr Glu Gly Gin He Leu Arg Asn Val He Lys Asp Tyr
1075 ' 1080 1085 TGG GGA .AAT CCT TTG AAG TTT GAT ACA GAA TAT TAT ATT ATT AAT GAT 3312 Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr He He Asn Asp 1090 ' 1095 1100
AAT TAT ATA GAT AGG TAT ATT GCA CCT GAA AGT AAT GTA CTT GTA CTT 3360 Asn Tyr He Asp Arg Tyr He Ala Pro Glu Ser Asn Val Leu Val Leu 1105 ' 1110 1115 1120
GTT CGG TAT CCA GAT AGA TCT AAA TTA TAT ACT GGA AAT CCT ATT ACT 3408 Val Arg Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro He Thr 1125 ' 1130 1135
ATT AAA TCA GTA TCT GAT AAG AAT CCT TAT AGT AGA ATT TTA AAT GGA 3456 He Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg He Leu Asn Gly 1140 1145 1150
GAT AAT ATA ATT CTT CAT ATG TTA TAT AAT AGT AGG AAA TAT ATG ATA 3504
Asp Asn He He Leu His Met Leu Tyr Asn Ser Arg Lys Tyr Met He 1155 1160 1165 ATA AGA GAT ACT GAT ACA ATA TAT GCA ACA CAA GGA GGA GAG TGT TCA 3552 He Arg Asp Thr Asp Thr He Tyr Ala Thr Gin Gly Gly Glu Cys Ser 1170 1175 1180
CAA AAT TGT GTA TAT GCA TTA AAA TTA CAG AGT AAT TTA GGT AAT TAT 3600 Gin Asn Cys Val Tyr Ala Leu Lys Leu Gin Ser Asn Leu Gly Asn Tyr 1185 1190 1195 1200
GGT ATA GGT ATA TTT AGT ATA AAA AAT ATT GTA TCT AAA AAT AAA TAT 3648 Gly He Gly He Phe Ser He Lys Asn He Val Ser Lys Asn Lys Tyr 1205 1210 ' 1215
TGT AGT CAA ATT TTC TCT AGT TTT AGG GAA AAT ACA ATG CTT CTA GCA 3696
Cys Ser Gin He Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu A a 1220 1225 1230
GAT ATA TAT AAA CCT TGG AGA TTT TCT TTT AAA AAT GCA TAC ACG CCA 3744
Asp He Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245 GTT GCA GTA ACT AAT TAT GAA ACA AAA CTA TTA TCA ACT TCA TCT TTT 3792 "al Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260
TGG AAA TTT ATT TCT AGG GAT CCA GGA TGG GTA GAG TAA 3831 Trp Lys Phe He Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 1275
(2) INFORMATION FOR SEQ ID NO.66 (l) SEQUENCE CHARACTERISTICS
(A) LENGTH 1276 amino acids
Figure imgf000350_0001
(D) TOPOLOGY linear Ui) MOLECULE TYPE protem
(/il SEQUENCE DESCRIPTION. SEQ ID NO 66
Met Thr Trp Pro Val Lys Asp Phe Aεn Tyr Ser Aεp Pro Val Asn Asp 1 b 10 15
Asn Asp He Leu Tyr Leu Arg He Pro Gin Asn Lys Leu He Thr Tnr 20 25 ' 30 Pro Val Lys Ala Phe Met He Thr Gin Asn He Trp Val He Pro Glu 35 40 45
Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lyε Pro Pro Arg Pro
Figure imgf000350_0002
Thr Ser Lys Tyr Gin Ser Tyr Tyr Asp Pro Ser Ivr Leu Ser rhr Asp 65 ' ' 70 75 80
Glu Gin Lys Asp Thr Phe Leu Lys Gly He He Lys Leu Phe Lyε Arg ' 85 90 95
He Asn Glu Arg Asp He Gly Lys Lys Leu He Aεn Tyr Leu Val Val
100 105 110
Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Aεp
115 120 125
Phe Thr Arg His Thr Thr Asn He Ala Val Glu Lys Phe Glu Asn Gly
130 135 140
Ser Trp Lys Val Thr Asn He He Thr Pro Ser Val Leu He Phe Gly
145 150 155 160
Pro Leu Pro Asn He Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gin Gly 165 170 175
Gin Gin Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser He Leu 180 185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Aεn 195 200 20 5
Gin Ser Ser Ala Val Leu Gly Lys Ser He Phe Cys Met Asp Pro Val 210 215 220
He Ala Leu Met His Glu Leu Thr His Ser Leu His Gin Leu Tyr Gly 225 230 235 240
He Asn He Pro Ser Asp Lys Arg He Arg Pro Gin Val Ser Glu Gly 245 250 255
Phe Phe Ser Gin Asp Gly Pro Asn Val Gin Phe Glu Glu Leu Tyr Thr 260 * 265 270 Phe Gly Gly Leu Asp Val Glu He He Pro Gin He Glu Arg Ser Gin 275 280 285
Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp He Ala Lys Arg Leu 290 295 300
Asn Asn He Asn Lys Thr He Pro Ser Ser Trp He Ser Asn He Asp 305 310 315 320
Lys Tyr Lys Lys He Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335
Thr Gly Asn Phe Val Val Asn He Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gin Tyr Asn 355 360 365
Val Lyε Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380
Ala Asn He Leu Asp Asp Asn He Tyr Thr He Arg Asp Gly Phe Asn 385 390 395 400
Leu Thr Aεn Lys Gly Phe Asn He Glu Asn Ser Gly Gin Asn He Glu ' 405 410 415
Arg Asn Pro Ala Leu Gin Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430 Pne Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445
Thr Cys He Lyε Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460
Asp Ser He Ser Gin Glu He Phe Glu Aεn Lys He He Thr Asp Glu 465 470 475 480
Thr Asn Val Gin Asn Tyr Ser Asp Asn Phe Ser Leu Asp Glu Ser He 485 490 495
Leu Aεp Gly Gin Val Pro He Asn Pro Glu He Val Asp Pro Leu Leu 500 505 510 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu He Val 515 520 525
Phe Tyr Asp Asp He Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr
530 535 540
Tyr Leu Glu Ser Gin Lys Leu Ser Asn Asn Val Glu Aεn He Thr Leu
545 550 555 560
Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys He Tyr Thr 565 570 575 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gin Ala Gly 580 585 590
Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605
He Met Lys Lys Asp Thr Leu Asp Lys He Ser Asp Val Ser Val He 610 615 620
He Pro Tyr He Gly Pro Ala Leu Asn He Gly Asn Ser Ala Leu Arg 625 630 635 640
Gly Asn Phe Lys Gin Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655
Glu Gly Phe Pro Glu Phe Thr He Pro Ala Leu Gly Val Phe Thr Phe 660 665 670
Tyr Ser Ser He Gin Glu Arg Glu Lys He He Lys Thr He Glu Asn 675 680 ' 685
Cys Leu Glu Gin Arg Val Lys Arg Trp Lys Asp Ser Tyr Gin Trp Met 690 695 700 Val Ser Asn Trp Leu Ser Arg He Thr Thr Gin Phe Asn His He Asn 705 710 715 720
Tyr Gin Met Tyr Asp Ser Leu Ser Tyr Gin Ala Asp Ala He Lys Ala 725 730 735
Lys He Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Aεp Lys Glu Asn 740 745 750
He Lys Ser Gin Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys He 755 760 765
Ser Glu Ala Met Asn Asn He Asn Lys Phe He Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val He Asp Glu Leu Asn 785 790 795 800
Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu He Asn Leu He Asp Ser 805 810 815
His Asn He He Leu Val Gly Glu Val Asp Arg Leu Ly: Ala Lys Val 820 825 830
Aεn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn He Phe Ser Tyr Thr 835 840 845
Asn Asn Ser Leu Leu Lys Asp He He Asn Glu Tyr Phe Asn Ser He 850 855 860 Asn Asp Ser Lys He Leu Ser Leu Gin Asn Lys Lys Asn Ala Leu Val 865 870 875 880
Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gin 885 890 895
Leu Asn Thr He Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910
Lys He He Val Asn Leu Asn Asn Asn He Leu Tyr Ser Ala He Tyr ' 915 920 925
Glu Asn Ser Ser Val Ser Phe Trp He Lys He Ser Lys Asp Leu Thr 930 935 940 Asn Ser His Asn Glu Tyr Thr He He Asn Ser He Glu Gin Asn Ser 945 950 955 960
Gly Trp Lys Leu Cys He Arg Asn Gly Asn He Glu Trp He Leu Gin 965 970 975
Asp Val Asn Arg Lys Tyr Lys Ser Leu He Phe Asp Tyr Ser Glu Ser 980 ' 985 990
Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr He Thr 995 1000 1005
Asn Asn He Met Gly Tyr Met Lys Leu Tyr He Asn Gly Glu Leu Lys 1010 1015 1020
Gin Ser Gin Lys He Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr 1025 1030 1035 1040
He Val Phe Gly He Asp Glu Asn He Asp Glu Asn Gin Met Leu Trp 1045 1050 1055
He Arg Asp Phe Asn He Phe Ser Lys Glu Leu Ser Asn Glu Asp He 1060 1065 1070
Aεn He Val Tyr Glu Gly Gin He Leu Arg Asn Val He Lys Asp Tyr 1075 1080 1085
Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr He He Asn Asp 1090 1095 ' 1100 Asn Tyr He Asp Arg Tyr He Ala Pro Glu Ser Asn Val Leu Val Leu 1105 1110 1115 1120
Val Arg Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro He Thr 1125 " 1130 1135
He Lys Ser Val Ser Asp Lys Aεn Pro Tyr Ser Arg He Leu Asn Gly 1140 1145 1150
Asp Asn He He Leu His Met Leu Tyr Asn Ser Arg Lys Tyr Met He 1155 1160 1165
He Arg Asp Thr Asp Thr He Tyr Ala Thr Gin Gly Gly Glu Cys Ser 1170 1175 1180 Gin Asn Cys Val Tyr Ala Leu Lys Leu Gin Ser Asn Leu Gly Asn Tyr 1185 ' 1190 1195 1200
Gly He Gly He Phe Ser He Lys Asn He Val Ser Lys Asn Lys Tyr 1205 1210 1215
Cys Ser Gin He Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala 1220 1225 1230
Asp He Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245
Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe 1250 1255 1260 Trp Lys Phe He Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 1275 (2) INFORMATION FOR SEQ ID NO: 67:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1469 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: DNA (genomic)
Hx) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 108..1460
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 67 :
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Figure imgf000354_0001
CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly Hiε He Glu Gly
5 10 15
CGT CAT ATG GCT AGC ATG GCT TTA TTA AAA GAT ATA ATT AAT GAA TAT 212 Arg Hiε Met Ala Ser Met Ala Leu Leu Lys Asp He He Asn Glu Tyr 20 25 30 35
TTC AAT AGT ATT AAT GAT TCA AAA ATT TTG AGC TTA CAA AAC AAA AAA 260 Phe Asn Ser He Asn Asp Ser Lys He Leu Ser Leu Gin Asn Lys Lys 40 45 50 AAT GCT TTA GTG GAT ACA TCA GGA TAT AAT GCA GAA GTG AGG GTA GGA 308 Asn Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly 55 60 65
GAT AAT GTT CAA CTT AAT ACG ATA TAT ACA AAT GAC TTT AAA TTA AGT 356 Asp Asn Val Gin Leu Asn Thr He Tyr Thr Asn Asp Phe Lys Leu Ser 70 75 80
AGT TCA GGA GAT AAA ATT ATA GTA AAT TTA AAT AAT AAT ATT TTA TAT 404 Ser Ser Gly Asp Lys He He Val Asn Leu Asn Asn Asn He Leu Tyr 85 90 95
AGC GCT ATT TAT GAG AAC TCT AGT GTT AGT TTT TGG ATT AAG ATA TCT 452 Ser Ala He Tyr Glu Asn Ser Ser Val Ser Phe Trp He Lys He Ser 100 ' 105 110 115
AAA GAT TTA ACT AAT TCT CAT AAT GAA TAT ACA ATA ATT AAC AGT ATA 500 Lys Asp Leu Thr Asn Ser His Aεn Glu Tyr Thr He He Asn Ser He 120 125 130 GAA CAA AAT TCT GGG TGG AAA TTA TGT ATT AGG AAT GGC AAT ATA GAA 548 Glu Gin Asn Ser Gly Trp Lys Leu Cys He Arg Asn Gly Asn He Glu 135 140 145
TGG ATT TTA CAA GAT GTT AAT AGA AAG TAT AAA AGT TTA ATT TTT GAT 596 Trp He Leu Gin Asp Val Aεn Arg Lys Tyr Lys Ser Leu He Phe Asp 150 155 160
TAT AGT GAA TCA TTA AGT CAT ACA GGA TAT ACA AAT AAA TGG TTT TTT 644 Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe 165 170 175
GTT ACT ATA ACT AAT AAT ATA ATG GGG TAT ATG AAA CTT TAT ATA AAT 692 Val Thr He Thr Aεn Aεn He Met Gly Tyr Met Lys Leu Tyr He Asn 180 185 190 ' 195 GGA GAA TTA AAG CAG AGT CAA AAA ATT GAA GAT TTA GAT GAG GTT AAG 740
Gly Glu Leu Lys Gin Ser Gin Lyε He Glu Asp Leu Asp Glu Val Lys 200 205 210 TTA GAT AAA ACC ATA GTA TTT GGA ATA GAT GAG AAT ATA GAT GAG AAT 788
Leu Asp Lys Thr He Val Phe Gly He Asp Glu Asn He Asp Glu Asn 215 220 225
CAG ATG CTT TGG ATT AGA GAT TTT AAT ATT TTT TCT AAA GAA TTA AGT 836 Gin Met Leu Trp He Arg Asp Phe Asn He Phe Ser Lys Glu Leu Ser 230 " 235 240
AAT GAA GAT ATT AAT ATT GTA TAT GAG GGA CAA ATA TTA AGA AAT GTT 884
Asn Glu Asp He Asn He Val Tyr Glu Gly Gin He Leu Arg Asn Val 245 250 255
ATT AAA GAT TAT TGG GGA AAT CCT TTG AAG TTT GAT ACA GAA TAT TAT 932
He Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr Tyr
260 265 270 275
ATT ATT AAT GAT AAT TAT ATA GAT AGG TAT ATT GCA CCT GAA AGT AAT 980
He He Asn Asp Asn Tyr He Asp Arg Tyr He Ala Pro Glu Ser Asn 280 285 290 GTA CTT GTA CTT GTT CGG TAT CCA GAT AGA TCT AAA TTA TAT ACT GGA 1028
'.'al Leu Val Leu Val Arg Tyr Pro Asp Arg Ser Lys Leu Tyr Thr Gly 295 300 305
.AAT CCT ATT ACT ATT AAA TCA GTA TCT GAT AAG AAT CCT TAT AGT AGA 1076 Asn Pro He Thr He Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg 310 ' 315 320
ATT TTA AAT GGA GAT AAT ATA ATT CTT CAT ATG TTA TAT AAT AGT AGG 1124
He Leu Asn Glv Asp Asn He He Leu His Met Leu Tyr Asn Ser Arg 325 ' 330 335
AAA TAT ATG ATA ATA AGA GAT ACT GAT ACA ATA TAT GCA ACA CAA GGA 1172
Lys Tyr Met He He Arg Asp Thr Asp Thr He Tyr Ala Thr Gin Gly
340 345 350 355
GGA GAG TGT TCA CAA AAT TGT GTA TAT GCA TTA AAA TTA CAG AGT AAT 1220
Gly Glu Cys Ser Gin Aεn Cys Val Tyr Ala Leu Lys Leu Gin Ser Aεn 360 365 370 TTA GGT AAT TAT GGT ATA GGT ATA TTT AGT ATA AAA AAT ATT GTA TCT 1268
Leu Glv Asn Tyr Gly He Gly He Phe Ser He Lys Asn He Val Ser 375 ' 380 385
AAA AAT AAA TAT TGT AGT CAA ATT TTC TCT AGT TTT AGG GAA AAT ACA 1316 Lys Asn Lys Tyr Cys Ser Gin He Phe Ser Ser Phe Arg Glu Asn Thr 390 395 400
ATG CTT CTA GCA GAT ATA TAT AAA CCT TGG AGA TTT TCT TTT AAA AAT 1364
Met Leu Leu Ala Asp He Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn 405 410 415
GCA TAC ACG CCA GTT GCA GTA ACT AAT TAT GAA ACA AAA CTA TTA TCA 1 12
Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser
420 425 430 435
ACT TCA TCT TTT TGG AAA TTT ATT TCT AGG GAT CCA GGA TGG GTA GAG 1460 Thr Ser Ser Phe Trp Lys Phe He Ser Arg Asp Pro Gly Trp Val Glu 440 445 450 TAAAAGCTT 1469
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 451 ammo acids ( B ) TYPE : amino acid ( D) TOPOLOGY : l inear
U i ) MOLECULE TYPE : protem
( κi ) SEQUENCE DESCRI PTION : SEQ ID NO : 68 :
Me t Gl y Hi s His His Hi s Hi s Hi s Hi s His Hi s Hi s Se r Ser Gly Hi s
1 5 10 15
He Glu Gly Arg His Met Ala Ser Met Ala Leu Leu Lys Asp He He 20 25 30
Asn Glu Tyr Phe Asn Ser He Asn Asp Ser Lys He Leu Ser Leu Gin 35 40 45
Asn Lys Lys Asn Ala Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val 50 55 60 Arg Val Gly Asp Asn Val Gin Leu Asn Thr He Tyr Thr Asn Asp Phe 65 ' 70 75 80
Lys Leu Ser Ser Ser Gly Asp Lys He He Val Asn Leu Asn Asn Asn 85 90 95
He Leu Tyr Ser Ala He Tyr Glu Asn Ser Ser Val Ser Phe Trp He 100 105 110
Lys He Ser Lys Asp Leu Thr Asn Ser His Asn Glu Tyr Thr He He 115 120 125
Asn Ser He Glu Gin Asn Ser Gly Trp Lys Leu Cys He Arg Asn Gly 130 135 140 Asn He Glu Trp He Leu Gin Asp Val Asn Arg Lyε Tyr Lys Ser Leu 145 150 155 160
He Phe Asp Tyr Ser Glu Ser Leu Ser His Thr Gly Tyr Thr Asn Lyε 165 170 175
Trp Phe Phe Val Thr He Thr Asn Asn He Met Gly Tyr Met Lys Leu 180 185 190
Tyr He Asn Gly Glu Leu Lys Gin Ser Gin Lys He Glu Asp Leu Asp 195 200 205
Glu Val Lys Leu Asp Lys Thr He Val Phe Gly He Asp Glu Asn He 210 " 215 220 Asp Glu Asn Gin Met Leu Trp He Arg Asp Phe Asn He Phe Ser Lys 225 230 235 240
Glu Leu Ser Asn Glu Aεp He Asn He Val Tyr Glu Gly Gin He Leu 245 250 255
Arg Asn Val He Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr 260 ' 265 270
Glu Tvr Tyr He He Asn Asp Asn Tyr He Asp Arg Tyr He Ala Pro ' 275 280 285
Glu Ser Asn Val Leu Val Leu Val Arg Tyr Pro Asp Arg Ser Lys Leu 290 295 300
Tyr Thr Glv Asn Pro He Thr He Lyε Ser Val Ser Asp Lys Asn Pro 305 310 315 320
Tyr Ser Arg He Leu Asn Gly Asp Asn He He Leu His Met Leu Tyr 325 330 335 Asn Ser Arg Lys Tyr Met He He Arg Asp Thr Asp Thr He Tyr Ala 340 345 350
Thr Gin Gly Gly Glu Cys Ser Gin Asn Cys Val Tyr Ala Leu Lys Leu 355 360 365
Gin Ser Asn Leu Gly Asn Tyr Gly He Gly He Phe Ser He Lys Asn 370 " 375 380 He Val Ser Lys Asn Lys Tyr Cys Ser Gin He Phe Ser Ser Phe Arg 385 ' 390 395 400
Glu Asn Thr Met Leu Leu Ala Asp He Tyr Lys Pro Trp Arg Phe Ser 405 410 415
Phe Lys Asn Ala Tyr Thr Pro Val Ala Val Thr Asn Tyr Glu Thr Lys 420 425 430
Leu Leu Ser Thr Ser Ser Phe Trp Lys Phe He Ser Arg Asp Pro Gly 435 440 445
Trp Val Glu 450 , INFORMATION FOR SEQ ID NO ■ 69
U) SEQUENCE CHARACTERISTICS
(A) LENGTH 32 base pairs
(B) TYPE nucleic acid (C) STRANDEDNESS single
(D) TOPOLOGY linear
Ui) MOLECULE TYPE other nucleic acid (A) DESCRIPTION /desc = "DNA"
(xi ) SEQUENCE DESCRIPTION SEQ ID NO 69
GCAAGCTTTT ACTCTACCCA TCCTGGATCC CT 32 2) INFORMATION FOR SEQ ID NO 70
U) SEQUENCE CHARACTERISTICS
(A) LENGTH 3825 base pairs
(B) TYPE nucleic acid (C) STRANDEDNESS double
(D) TOPOLOGY linear
Ui) MOLECULE TYPE DNA (genomic) (ix) FEATURE
(A) NAME/KEY CDS
(B) LOCATION 1 3822
(xi) SEQUENCE DESCRIPTION SEQ ID NO 70
ATG CCA GTT GCA ATA AAT AGT TTT AAT TAT AAT GAC CCT GTT AAT GAT 48
Met Pro Val Ala He Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 GAT ACA ATT TTA TAC ATG CAG ATA CCA TAT GAA GAA AAA AGT AAA AAA 96
Asp Thr He Leu Tyr Met Gin He Pro Tyr Glu Glu Lys Sei Lys Lys 20 25 " 30
TAT TAT AAA GCT TTT GAG ATT ATG CGT AAT GTT TGG ATA ATT CCT GAG 144 Tyr Tyr Lys Ala Phe Glu He Met Arg Asn Val Trp He He Pro Glu 35 40 45
AGA AAT ACA ATA GGA ACG AAT CCT AGT GAT TTT GAT CCA CCG GCT TCA 192 Arg Asn Thr He Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60 TTA AAG AAC GGA AGC AGT GCT TAT TAT GAT CCT AAT TAT TTA ACC ACT 240 Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 GAT GCT GAA AAA GAT AGA TAT TTA AAA ACA ACG ATA AAA TTA TTT AAG 288 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr He Lys Leu Phe Lys 85 90 95
AGA ATT AAT AGT AAT CCT GCA GGG AAA GTT TTG TTA CAA GAA ATA TCA 336 Arg He Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gin Glu He Ser
100 105 110
TAT GCT AAA CCA TAT TTA GGA AAT GAC CAC ACG CCA ATT GAT GAA TTC 384 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro He Asp Glu Phe 115 120 125
TCT CCA GTT ACT AGA ACT ACA AGT GTT AAT ATA AAA TTA TCA ACT AAT 432
Ser Pro Val Thr Arg Thr Thr Ser Val Asn He Lys Leu Ser Thr Asn 130 135 140
GTT GAA AGT TCA ATG TTA TTG AAT CTT CTT GTA TTG GGA GCA GGA CCT 480
Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro
145 150 155 * 160 GAT ATA TTT GAA AGT TGT TGT TAC CCC GTT AGA AAA CTA ATA GAT CCA 528 Asp He Phe Glu Ser Cys Cys Tyr Pro Val Arg Lyε Leu He Asp Pro 165 170 175
GAT GTA GTT TAT GAT CCA AGT AAT TAT GGT TTT GGA TCA ATT AAT ATC 576 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser He Asn He
180 185 190
GTG ACA TTT TCA CCT GAG TAT GAA TAT ACT TTT AAT GAT ATT AGT GGA 624 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp He Ser Gly 195 200 205
GGG CAT AAT AGT AGT ACA GAA TCA TTT ATT GCA GAT CCT GCA ATT TCA 672 Gly His Asn Ser Ser Thr Glu Ser Phe He Ala Asp Pro Ala He Ser 210 215 220
CTA GCT CAT GAA TTG ATA CAT GCA CTG CAT GGA TTA TAC GGG GCT AGG 720
Leu Ala His Glu Leu He His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240 GGA GTT ACT TAT GAA GAG ACT ATA GAA GTA AAG CAA GCA CCT CTT ATG 768 Glv Val Thr Tyr Glu Glu Thr He Glu Val Lys Gin Ala Pro Leu Met 245 250 255
ATA GCC GAA AAA CCC ATA AGG CTA GAA GAA TTT TTA ACC TTT GGA GGT 816 He Ala Glu Lys Pro He Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly
260 265 270
CAG GAT TTA AAT ATT ATT ACT AGT GCT ATG AAG GAA AAA ATA TAT AAC 864 Gin Aεp Leu Asn He He Thr Ser Ala Met Lys Glu Lys He Tyr Asn 275 280 285
AAT CTT TTA GCT AAC TAT GAA AAA ATA GCT ACT AGA CTT AGT GAA GTT 912 Asn Leu Leu Ala Asn Tyr Glu Lys He Ala Thr Arg Leu Ser Glu Val 290 295 300
AAT AGT GCT CCT CCT GAA TAT GAT ATT AAT GAA TAT AAA GAT TAT TTT 960 Aεn Ser Ala Pro Pro Glu Tyr Asp He Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320 CAA TGG AAG TAT GGG CTA GAT AAA AAT GCT GAT GGA AGT TAT ACT GTA 1008 Gin Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335
AAT GAA AAT AAA TTT AAT GAA ATT TAT AAA AAA TTA TAT AGT TTT ACA 1056 Asn Glu Asn Lys Phe Asn Glu He Tyr Lys Lys Leu Tyr Ser Phe Thr
>56 340 345 350
GAG AGT GAC TTA GCA AAT AAA TTT AAA GTA AAA TGT AGA AAT ACT TAT 1104
Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365
TTT ATT AAA TAT GAA TTT TTA AAA GTT CCA AAT TTG TTA GAT GAT GAT 1152
Phe He Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380
ATT TAT ACT GTA TCA GAG GGG TTT AAT ATA GGT AAT TTA GCA GTA AAC 1200
He Tyr Thr Val Ser Glu Gly Phe Asn He Gly Asn Leu Ala Val Asn 385 390 395 400 AAT CGC GGA CAA AGT ATA AAG TTA AAT CCT AAA ATT ATT GAT TCC ATT 1248
Asn Arg Gly Gin Ser He Lys Leu Asn Pro Lys He He Asp Ser He
405 410 415
CCA GAT AAA GGT CTA GTA GAA AAG ATC GTT AAA TTT TGT AAG AGC GTT 1296 Pro Aεp Lys Gly Leu Val Glu Lys He Val Lys Phe Cys Lys Ser Val
420 425 430
ATT CCT AGA AAA GGT ACA AAG GCG CCA CCG CGA CTA TGC ATT AGA GTA 1344
He Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys He Arg Val 435 440 445
AAT AAT AGT GAG TTA TTT TTT GTA GCT TCA GAA AGT AGC TAT AAT GAA 1392
Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455 460
AAT GAT ATT AAT ACA CCT AAA GAA ATT GAC GAT ACA ACA AAT CTA AAT 1440
Asn Asp He Asn Thr Pro Lys Glu He Asp Asp Thr Thr Asn Leu Asn 465 470 475 480 AAT AAT TAT AGA AAT AAT TTA GAT GAA GTT ATT TTA GAT TAT AAT AGT 1488
Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val He Leu Asp Tyr Asn Ser
485 490 495
CAG ACA ATA CCT CAA ATA TCA AAT CGA ACA TTA AAT ACA CTT GTA CAA 1536 Gin Thr He Pro Gin He Ser Aεn Arg Thr Leu Asn Thr Leu Val Gin
500 505 510
GAC AAT AGT TAT GTG CCA AGA TAT GAT TCT AAT GGA ACA AGT GAA ATA 1584
Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu He 515 520 525
GAG GAA TAT GAT GTT GTT GAC TTT AAT GTA TTT TTC TAT TTA CAT GCA 1632
Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540
CAA AAA GTG CCA GAA GGT GAA ACC AAT ATA AGT TTA ACT TCT TCA ATT 1680
Gin Lys Val Pro Glu Gly Glu Thr Asn He Ser Leu Thr Ser Ser He 545 550 555 560 GAT ACA GCA TTA TTA GAA GAA TCC AAA GAT ATA TTT TTT TCT TCA GAG 1728
Aεp Thr Ala Leu Leu Glu Glu Ser Lys Asp He Phe Phe Ser Ser Glu
565 570 575
TTT ATC GAT ACT ATC AAT AAA CCT GTA AAT GCA GCA CTA TTT ATA GAT 1776 Phe He Asp Thr He Asn Lys Pro Val Asn Ala Ala Leu Phe He Asp
580 585 590
TGG ATA AGC AAA GTA ATA AGA GAT TTT ACC ACT GAA GCT ACA CAA AAA 1824
Trp He Ser Lys Val He Arg Asp Phe Thr Thr Glu Ala Thr Gin Lys 595 600 605
AGT ACT GTT GAT AAG ATT GCA GAC ATA TCT TTA ATT GTA CCC TAT GTA 1872
Ser Thr Val Asp Lys He Ala Asp He Ser Leu He Val Pro Tyr Val 610 615 620 GGT CTT GCT TTG AAT ATA ATT ATT GAG GCA GAA AAA GGA AAT TTT GAG 1920 Gly Leu Ala Leu Asn He He He Glu Ala Glu Lys Gly Asn Phe Glu 625 630 635 640
GAG GCA TTT GAA TTA TTA GGA GTG GGT ATT TTA TTA GAA TTT GTG CCA 1968 Glu Ala Phe Glu Leu Leu Gly Val Gly He Leu Leu Glu Phe Val Pro 645 650 655
GAA CTT ACA ATT CCT GTA ATT TTA GTG TTT ACG ATA AAA TCC TAT ATA 2016 Glu Leu Thr He Pro Val He Leu Val Phe Thr He Lys Ser Tyr He 660 665 670
GAT TCA TAT GAG AAT AAA AAT AAA GCA ATT AAA GCA ATA AAT AAT TCA 2064 Asp Ser Tyr Glu Asn Lys Asn Lys Ala He Lys Ala He Asn Asn Ser 675 680 685
TTA ATC GAA AGA GAA GCA AAG TGG AAA GAA ATA TAT AGT TGG ATA GTA 2112 Leu He Glu Arg Glu Ala Lys Trp Lys Glu He Tyr Ser Trp He Val 690 695 700
TCA AAT TGG CTT ACT AGA ATT AAT ACT CAA TTT AAT AAA AGA AAA GAG 2160 Ser Asn Trp Leu Thr Arg He Asn Thr Gin Phe Asn Lys Arg Lys Glu 705 710 715 720 CAA ATG TAT CAG GCT TTA CAA AAT CAA GTA GAT GCA ATA AAA ACA GCA 2208 Gin Met Tyr Gin Ala Leu Gin Asn Gin Val Asp Ala He Lys Thr Ala 725 730 735
ATA GAA TAT AAA TAT AAT AAT TAT ACT TCA GAT GAG AAA AAT AGA CTT 2256 He Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu
740 745 750
GAA TCT GAA TAT AAT ATC AAT AAT ATA GAA GAA GAA TTG AAT AAA AAA 2304 Glu Ser Glu Tyr Asn He Asn Asn He Glu Glu Glu Leu Asn Lys Lys 755 760 765
GTT TCT TTA GCA ATG AAA AAT ATA GAA AGA TTT ATG ACA GAA AGT TCT 2352
Val Ser Leu Ala Met Lys Asn He Glu Arg Phe Met Thr Glu Ser Ser
770 775 780
ATA TCT TAT TTA ATG AAA TTA ATA AAT GAA GCC AAA GTT GGT AAA TTA 2400
He Ser Tyr Leu Met Lys Leu He Asn Glu Ala Lyε Val Gly Lys Leu
785 790 795 800 AAA AAA TAT GAT AAC CAT GTT AAG AGC GAT TTA TTA AAC TAT ATT CTC 2448 Lvs Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr He Leu 805 810 815
GAC CAT AGA TCA ATC TTA GGA GAG CAG ACA AAT GAA TTA AGT GAT TTG 2496 Asp His Arg Ser He Leu Gly Glu Gin Thr Asn Glu Leu Ser Asp Leu
820 825 830
GTG ACT AGT ACT TTG AAT AGT AGT ATT CCA TTT GAA CTT TCT TCA TAT 2544 Val Thr Ser Thr Leu Asn Ser Ser He Pro Phe Glu Leu Ser Ser Tyr 835 840 845
ACT AAT GAT AAA ATT CTA ATT ATA TAT TTT AAT AGA TTA TAT AAA AAA 2592 Thr Asn Asp Lys He Leu He He Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860
ATT AAA GAT AGT TCT ATT TTA GAT ATG CGA TAT GAA AAT AAT AAA TTT 2640 He Lys Asp Ser Ser He Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 865 870 875 ' 880 ATA GAT ATC TCT GGA TAT GGT TCA AAT ATA AGC ATT AAT GGA AAC GTA 2688 He Asp He Ser Gly Tyr Gly Ser Asn He Ser He Asn Gly Asn Val 885 890 895 TAT ATT TAT TCA ACA AAT AGA AAT CAA TTT GGA ATA TAT AAT AGT AGG 2736 Tyr He Tyr Ser Thr Asn Arg Asn Gin Phe Gly He Tyr Asn Ser Arg 900 905 910
CTT AGT GAA GTT AAT ATA GCT CAA AAT AAT GAT ATT ATA TAC AAT AGT 2784 Leu Ser Glu Val Asn He Ala Gin Asn Asn Asp He He Tyr Asn Ser 915 920 925
AGA TAT CAA AAT TTT AGT ATT AGT TTC TGG GTA AGG ATT CCT AAA CAC 2832 Arg Tyr Gin Asn Phe Ser He Ser Phe Trp Val Arg He Pro Lys His 930 935 940
TAC AAA CCT ATG AAT CAT AAT CGG GAA TAC ACT ATA ATA AAT TGT ATG 2880
Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr He He Asn Cys Met 945 950 955 960
GGG AAT AAT AAT TCG GGA TGG AAA ATA TCA CTT AGA ACT GTT AGA GAT 2928
Gly Asn Asn Asn Ser Gly Trp Lys He Ser Leu Arg Thr Val Arg Asp 965 970 975 TGT GAA ATA ATT TGG ACT TTA CAA GAT ACT TCT GGA AAT AAG GAA AAT 2976 Cys Glu He He Trp Thr Leu Gin Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990
TTA ATT TTT AGG TAT GAA GAA CTT AAT AGG ATA TCT AAT TAT ATA AAT 3024 Leu He Phe Arg Tyr Glu Glu Leu Asn Arg He Ser Asn Tyr He Asn 995 ' 1000 1005
.AAA TGG ATT TTT GTA ACT ATT ACT AAT AAT AGA TTA GGC AAT TCT AGA 3072 Lvs Trp He Phe Val Thr He Thr Asn Asn Arg Leu Gly Asn Ser Arg L010 1015 1020
ATT TAC ATC AAT GGA AAT TTA ATA GTT GAA AAA TCA ATT TCG AAT TTA 120 He Tyr He Asn Gly Asn Leu He Val Glu Lys Ser He Ser Asn Leu 1025 1030 1035 1040
GGT GAT ATT CAT GTT AGT GAT AAT ATA TTA TTT AAA ATT GTT GGT TGT 3168 Gly Aεp He His Val Ser Asp Asn He Leu Phe Lys He Val Gly Cyε 1045 1050 1055 GAT GAT GAA ACG TAT GTT GGT ATA AGA TAT TTT AAA GTT TTT AAT ACG 3216 Asp Asp Glu Thr Tvr Val Glv He Arg Tyr Phe Lys Val Phe Aεn Thr 1060 ' ' 1065 ' 1070
GAA TTA GAT AAA ACA GAA ATT GAG ACT TTA TAT AGT AAT GAG CCA GAT 3264 Glu Leu Asp Lys Thr Glu He Glu Thr Leu Tyr Ser Asn Glu Pro Aεp 1075 1080 1085
CCA AGT ATC TTA AAA AAC TAT TGG GGA AAT TAT TTG CTA TAT AAT AAA 3312 Pro Ser He Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lys 1090 1095 1100
AAA TAT TAT TTA TTC AAT TTA CTA AGA AAA GAT AAG TAT ATT ACT CTG 3360 Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr He Thr Leu 1105 1110 1115 1120
AAT TCA GGC ATT TTA AAT ATT AAT CAA CAA AGA GGT GTT ACT GAA GGC 3408 Asn Ser Gly He Leu Asn He Asn Gin Gin Arg Gly Val Thr Glu Gly 1125 1130 1135 TCT GTT TTT TTG AAC TAT AAA TTA TAT GAA GGA GTA GAA GTC ATT ATA 3456 Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val He He 1140 1145 1150
AGA AAA AAT GGT CCT ATA GAT ATA TCT AAT ACA GAT AAT TTT GTT AGA 3504 Arg Lys Asn Gly Pro He Asp He Ser Asn Thr Asp Asn Phe Val Arg 1155 1160 1165
AAA AAC GAT CTA GCA TAC ATT AAT GTA GTA GAT CGT GGT GTA GAA TAT 3552 Lys Asn Asp Leu Ala Tyr He Asn Val Val Asp Arg Gly Val Glu Tyr 1170 1175 1180
CGG TTA TAT GCT GAT ACA AAA TCA GAG AAA GAG AAA ATA ATA AGA ACA 3600 Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys He He Arg Thr 1185 1190 1195 " 1200
TCT AAT CTA AAC GAT AGC TTA GGT CAA ATT ATA GTT ATG GAT TCA ATA 3648 Ser Asn Leu Asn Asp Ser Leu Gly Gin He He Val Met Asp Ser He 1205 1210 1215 GGA AAT AAT TGC ACA ATG AAT TTT CAA AAC AAT AAT GGG AGC AAT ATA 3696 Gly Asn Asn Cys Thr Met Asn Phe Gin Asn Asn Asn Gly Ser Asn He 1220 1225 1230
GGA TTA CTA GGT TTT CAT TCA AAT AAT TTG GTT GCT AGT AGT TGG TAT 3744 Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245
TAT AAC AAT ATA CGA AGA AAT ACT AGC AGT AAT GGA TGC TTT TGG AGT 3792 Tyr Asn Asn He Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260
TCT ATT TCT AAA GAG AAT GGA TGG AAA GAA TGA 3825
Ser He Ser Lys Glu Aεn Gly Trp Lys Glu 1265 1270
(2) INFORMATION FOR SEQ ID NO : 71 :
U) SEQUENCE CHARACTERISTICS.
(A) LENGTH: 1274 ammo acids (B) TYPE: amino acid
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO .71 ■
Met Pro Val Ala He Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 10 15 Asp Thr He Leu Tyr Met G n He Pro Tyr Glu Glu Lys Ser Lys Lyε
20 25 30
Tyr Tyr Lys Ala Phe Glu He Met Arg Asn Val Trp He He Pro Glu 35 40 45
Aig Asn Thr He Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60
Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80
Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr He Lyε Leu Phe Lys 85 90 95 Arg He Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gin Glu He Ser
100 105 110
Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro He Asp Glu Phe
115 120 125
Ser Pro Val Thr Arg Thr Thr Ser Val Asn He Lys Leu Ser Thr Asn
130 135 140
Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160 Asp He Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu He Asp Pro 165 170 175
Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser He Asn He 180 185 ' 190
Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp He Ser Gly 195 200 205 Gly His Asn Ser Ser Thr Glu Ser Phe He Ala Asp Pro Ala He Ser 210 215 220
Leu Ala His Glu Leu He His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240
Gly Val Thr Tyr Glu Glu Thr He Glu Val Lys Gin Ala Pro Leu Met 245 250 255
He Ala Glu Lys Pro He Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270
Gin Asp Leu Aεn He He Thr Ser Ala Met Lys Glu Lys He Tyr Asn 275 280 285 Asn Leu Leu Ala Asn Tyr Glu Lys He Ala Thr Arg Leu Ser Glu Val 290 295 300
Asn Ser Ala Pro Pro Glu Tyr Asp lie Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320
Gin Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 ' 335
Asn Glu Asn Lys Phe Asn Glu He Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350
51 u Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cyε Arg Asn Thr Tyr 355 360 365 Phe He Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 ' 375 380
He Tyr Thr Val Ser Glu Gly Phe Asn He Gly Asn Leu Ala Val Asn
385 390 395 400
Aεn Arg Gly Gin Ser He Lyε Leu Aεn Pro Lyε He He Aεp Ser He
405 410 415
Pro Asp Lyε Gly Leu Val Glu Lys He Val Lys Phe Cys Lys Ser Val 420 425 430
He Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys He Arg Val
435 440 445 Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu
450 455 460
Asn Asp He Asn Thr Pro Lys Glu He Asp Asp Thr Thr Asn Leu Asn
465 470 475 480
Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val He Leu Asp Tyr Asn Ser
485 490 495
Gin Thr He Pro Gin He Ser Asn Arg Thr Leu Asn Thr Leu Val Gin 500 505 510
Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu He 515 520 525 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540
Gin Lys Val Pro Glu Gly Glu Thr Asn He Ser Leu Thr Ser Ser He 545 550 555 560
Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp He Phe Phe Ser Ser Glu 565 570 575
Phe He Asp Thr He Asn Lys Pro Val Asn Ala Ala Leu Phe He Asp 580 585 590
Trp He Ser Lys Val He Arg Asp Phe Thr Thr Glu Ala Thr G.ln Lys 595 600 605 Ser Thr Val Asp Lys He Ala Asp He Ser Leu He Val Pro Tyr Val 610 615 620
Gly Leu Ala Leu Asn He He He Glu Ala Glu Lys Gly Asn Phe Glu 625 630 635 640
Glu Ala Phe Glu Leu Leu Gly Val Gly He Leu Leu Glu Phe Val Pro 645 650 655
Glu Leu Thr He Pro Val He Leu Val Phe Thr He Lys Ser Tyr He 660 665 670
Asp Ser Tyr Glu Asn Lys Asn Lys Ala He Lys Ala He Asn Asn Ser 675 680 685 Leu He Glu Arg Glu Ala Lys Trp Lys Glu He Tyr Ser Trp He Val 690 695 ' 700
Ser Asn Trp Leu Thr Arg He Asn Thr Gin Phe Asn Lys Arg Lys Glu 705 710 715 720
Gin Met Tyr Gin Ala Leu Gin Asn Gin Val Asp Ala He Lys Thr Ala 725 730 735
He Glu Tyr Lyε Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750
Glu Ser Glu Tyr Asn He Asn Asn He Glu Glu Glu Leu Asn Lyε Lys
755 760 765 Val Ser Leu Ala Met Lys Asn He Glu Arg Phe Met Thr Glu Ser Ser
770 775 780
He Ser Tyr Leu Met Lys Leu He Asn Glu Ala Lys Val Gly Lys Leu 785 " 790 795 800
Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr He Leu 805 ' 810 ' 815
Asp His Arg Ser He Leu Gly Glu Gin Thr Asn Glu Leu Ser Asp Leu 820 825 830
Val Thr Ser Thr Leu Asn Ser Ser He Pro Phe Glu Leu Ser Ser Tyr
835 840 845 Thr Asn Asp Lys He Leu He He Tyr Phe Asn Arg Leu Tyr Lys Lys
850 ' 855 860
He Lys Asp Ser Ser He Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 865 870 875 880
He Asp He Ser Gly Tyr Gly Ser Asn He Ser He Asn Gly Asn Val 885 890 895
Tyr He Tyr Ser Thr Asn Arg Asn Gin Phe Gly He Tyr Asn Ser Arg 900 905 910 Leu Ser Glu Val Asn He Ala Gin Asn Asn Asp He He Tyr Asn Ser
915 920 925
Arg Tyr Gin Asn Phe Ser He Ser Phe Trp Val Arg He Pro Lys His 930 935 940
Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr He He Asn Cys Met 945 950 955 960 Gly Asn Asn Asn Ser Gly Trp Lys He Ser Leu Arg Thr Val Arg Asp
965 970 975
Cys Glu He He Trp Thr Leu Gin Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990
Leu He Phe Arg Tyr Glu Glu Leu Asn Arg He Ser Asn Tyr He Asn 995 1000 1005
Lys Trp He Phe Val Thr He Thr Asn Asn Arg Leu Gly Asn Ser Arg 1010 1015 1020
He Tyr He Asn Gly Asn Leu He Val Glu Lys Ser He Ser Asn Leu 1025 1030 1035 1040
Gly Asp He His Val Ser Asp Asn He Leu Phe Lys He Val Gly Cys 1045 1050 1055
Asp Asp Glu Thr Tyr Val Gly He Arg Tyr Phe Lys Val Phe Aεn Thr 1060 1065 ' ' 1070
Glu Leu Asp Lys Thr Glu He Glu Thr Leu Tyr Ser Asn Glu Pro Asp 1075 1080 1085
Pro Ser He Leu Lyε Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr Asn Lyε
1090 1095 1100
Lys T' 7yr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr He Thr reu 1105 1110 1115 1120 Asn Ser Gly He Leu Asn He Asn Gin Gin Arg Gly Val Thr Glu Gly
1125 1130 1135
Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val He He 1140 1145 1150
Arg Lys Aεn Gly Pro He Aεp He Ser Asn Thr Asp Aεn Phe Val Arg 1155 1160 1165
Lys Asn Asp Leu Ala Tyr He Aεn Val Val Aεp Arg Gly Val Glu lyr 1170 1175 1180
Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lys He He Arg Thr 1185 1190 1195 1200 Ser Asn Leu Asn Asp Ser Leu Gly Gin He He Val Met Asp Ser He
1205 1210 1215
Gly Asn Asn Cys Thr Met Asn Phe Gin Asn Asn Asn Gly Ser Asn He 1220 1225 1230
Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245
Tyr Asn Asn He Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp Ser 1250 1255 1260
Ser He Ser Lys Glu Asn Gly Trp Lys Glu
1265 1270 (2) INFORMATION FOR SEQ ID NO: 72. ( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH : 1460 base pai rs
( B ) TYPE : nucleic acid
( C ) STRANDEDNESS : double
( D) TOPOLOGY : l inear
Ui) MOLECULE TYPE: DNA (genomic)
Ux) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 108..1451
(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Met Gly His
1
CA.T CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His Hiε His His His His Ser Ser Gly His He Glu Gly 5 10 15 CGT CAT ATG GCT AGC ATG GCT ATT CTA ATT ATA TAT TTT AAT AGA TTA 212 Arg His Met Ala Ser Met Ala He Leu He He Tyr Phe Asn Arg Leu 20 25 30 " 35
TAT AAA AAA ATT AAA GAT AGT TCT ATT TTA GAT ATG CGA TAT GAA AAT 260 Tyr Lys Lys He Lys Asp Ser Ser He Leu Asp Met Arg Tyr Glu Asn
40 45 50
AAT AAA TTT ATA GAT ATC TCT GGA TAT GGT TCA AAT ATA AGC ATT AAT 308 Asn Lys Phe He Asp He Ser Gly Tyr Gly Ser Asn He Ser He Asn ' 55 60 65
GGA AAC GTA TAT ATT TAT TCA ACA AAT AGA AAT CAA TTT GGA ATA TAT 356
Gly Asn Val Tyr He Tyr Ser Thr Asn Arg Asn Gin Phe Gly He Tyr
70 75 80
AAT AGT AGG CTT AGT GAA GTT AAT ATA GCT CAA AAT AAT GAT ATT ATA 404 Asn Ser Arg Leu Ser Glu Val Asn He Ala Gin Asn Asn Asp He He 85 90 95 TAC AAT AGT AGA TAT CAA AAT TTT AGT ATT AGT TTC TGG GTA AGG ATT 452 Tyr Asn Ser Arg Tyr Gin Asn Phe Ser He Ser Phe Trp Val Arg He 100 ' 105 110 115
CCT AAA CAC TAC AAA CCT ATG AAT CAT AAT CGG GAA TAC ACT ATA ATA 500 Pro Lys His Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr He He
120 125 130
AAT TGT ATG GGG AAT AAT AAT TCG GGA TGG AAA ATA TCA CTT AGA ACT 548 Asn Cys Met Gly Asn Asn Asn Ser Gly Trp Lys He Ser Leu Arg Thr 135 140 145
GTT AGA GAT TGT GAA ATA ATT TGG ACT TTA CAA GAT ACT TCT GGA AAT 596
Val Arg Asp Cys Glu He He Trp Thr Leu Gin Asp Thr Ser Gly Asn 150 155 160
AAG GAA AAT TTA ATT TTT AGG TAT GAA GAA CTT AAT AGG ATA TCT AAT 644
Lys Glu Asn Leu He Phe Arg Tyr Glu Glu Leu Asn Arg He Ser Asn
165 170 175 TAT ATA AAT AAA TGG ATT TTT GTA ACT ATT ACT AAT AAT AGA TTA GGC 692 Tyr He Asn Lys Trp He Phe Val Thr He Thr Asn Asn Arg Leu Gly 180 185 190 195
AAT TCT AGA ATT TAC ATC AAT GGA AAT TTA ATA GTT GAA AAA TCA ATT 740 Asn Ser Arg He Tyr He Asn Gly Asn Leu He Val Glu Lys Ser He 200 205 210
TCG AAT TTA GGT GAT ATT CAT GTT AGT GAT AAT ATA TTA TTT AAA ATT 788 Ser Asn Leu Gly Asp He His Val Ser Asp Asn He Leu Phe Lys He 215 220 225
GTT GGT TGT GAT GAT GAA ACG TAT GTT GGT ATA AGA TAT TTT AAA GTT 836 Val Gly Cys Asp Asp Glu Thr Tyr Val Gly He Arg Tyr Phe Lys Val 230 235 240
TTT AAT ACG GAA TTA GAT AAA ACA GAA ATT GAG ACT TTA TAT AGT AAT 884 Phe Asn Thr Glu Leu Asp Lys Thr Glu He Glu Thr Leu Tyr Ser Aεn 245 250 255 GAG CCA GAT CCA AGT ATC TTA AAA AAC TAT TGG GGA AAT TAT TTG CTA 932 Glu Pro Asp Pro Ser He Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu 260 265 270 275
TAT AAT AAA AAA TAT TAT TTA TTC AAT TTA CTA AGA AAA GAT AAG TAT 980 Tyr Asn Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp Lys Tyr
280 285 290
ATT ACT CTG AAT TCA GGC ATT TTA AAT ATT AAT CAA CAA AGA GGT GTT 1028 He Thr Leu Asn Ser Gly He Leu Asn He Asn Gin Gin Arg Gly Val 295 300 305
ACT G.AA GGC TCT GTT TTT TTG AAC TAT AAA TTA TAT GAA GGA GTA GAA 1076
Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu Gly Val Glu 310 315 320
GTC ATT ATA AGA AAA AAT GGT CCT ATA GAT ATA TCT AAT ACA GAT AAT 1124
Val He He Arg Lys Asn Gly Pro He Asp He Ser Asn Thr Asp Asn 325 330 335 TTT GTT AGA AAA AAC GAT CTA GCA TAC ATT AAT GTA GTA GAT CGT GGT 1172 Phe Val Arg Lys Asn Asp Leu Ala Tyr He Asn Val Val Asp Arg Gly 340 345 350 355
GTA GAA TAT CGG TTA TAT GCT GAT ACA AAA TCA GAG AAA GAG AAA ATA 1220 Val Glu Tyr Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu Lyε He
360 365 370
ATA AGA ACA TCT AAT CTA AAC GAT AGC TTA GGT CAA ATT ATA GTT ATG 1268 He Arg Thr Ser Asn Leu Asn Asp Ser Leu Gly Gin He He Val Met 375 380 385
GAT TCA ATA GGA AAT AAT TGC ACA ATG AAT TTT CAA AAC AAT AAT GGG 1316
Asp Ser He Gly Asn Asn Cys Thr Met Asn Phe Gin Asn Asn Asn Gly
390 395 400
AGC AAT ATA GGA TTA CTA GGT TTT CAT TCA AAT AAT TTG GTT GCT AGT 1364
Ser Asn He Gly Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser
405 * 410 415 AGT TGG TAT TAT AAC AAT ATA CGA AGA AAT ACT AGC AGT AAT GGA TGC 1412 Ser Trp Tyr Tyr Asn Asn He Arg Arg Asn Thr Ser Ser Asn Gly Cys 420 425 430 435
TTT TGG AGT TCT ATT TCT AAA GAG AAT GGA TGG AAA GAA TGAAAGCTT 1460 Phe Trp Ser Ser He Ser Lys Glu Asn Gly Trp Lys Glu
440 445
(2) INFORMATION FOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 448 ammo acids
Figure imgf000367_0001
(D) TOPOLOGY: linear Ui) MOLECULE TYPE: protem (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Met Gly His His His His His His His His His His Ser Ser Gly His
1 5 10 15
5
He Glu Gly Arg His Met Ala Ser Met Ala He Leu He He Tyr Phe 20 25 30
Asn Arg Leu Tyr Lys Lys He Lys Asp Ser Ser He Leu Asp Met Arg 10 35 40 45
Tyr Glu Asn Asn Lys Phe He Asp He Ser Gly Tyr Gly Ser Asn He 50 55 60
15 Ser He Aεn Gly Asn Val Tyr He Tyr Ser Thr Aεn Arg Aεn Gin Phe 65 70 75 80
Gly He Tyr Asn Ser Arg Leu Ser Glu Val Asn He Ala Gin Asn Asn 85 90 95
20
Asp He He Tyr Asn Ser Arg Tyr Gin Asn Phe Ser He Ser Phe Trp 100 105 110
Val Arg He Pro Lys His Tyr Lys Pro Met Asn His Asn Arg Glu Tyr
">> 115 120 125
Thr He He Asn Cys Met Gly Asn Asn Asn Ser Gly Trp Lys He Ser 130 135 140
30 Leu Arg Thr Val Arg Asp Cys Glu He He Trp Thr Leu Gin Asp Thr 145 150 155 160
Ser Gly Asn Lys Glu Asn Leu He Phe Arg Tyr Glu Glu Leu Asn Arg 165 170 175
He Ser Asn Tyr He Asn Lys Trp He Phe Val Thr He Thr Asn Asn 180 185 190
Arg Leu Gly Asn Ser Arg He Tyr He Asn Gly Asn Leu He Val Glu 40 195 200 205
Lys Ser He Ser Asn Leu Gly Asp He His Val Ser Asp Asn He Leu 210 215 220
45 Phe Lvs He Val Gly Cys Asp Asp Glu Thr Tyr Val Gly He Arg Tyr 225 230 235 240
Phe Lys Val Phe Asn Thr Glu Leu Asp Lys Thr Glu He Glu Thr Leu
245 250 255
50
Tyr Ser Asn Glu Pro Asp Pro Ser He Leu Lys Asn Tyr Trp Gly Asn
260 265 270
Tyr Leu Leu Tyr Asn Lys Lyε Tyr Tyr Leu Phe Asn Leu Leu Arg Lys
55 275 2B0 285
Asp Lys Tyr He Thr Leu Asn Ser Gly He Leu Asn He Asn Gin Gin
290 295 300
60 Arg Gly Val Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys Leu Tyr Glu
305 310 315 320
Gly Val Glu Val He He Arg Lys Asn Gly Pro He Asp He Ser Asn 325 330 " 335
65
Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr He Asn Val Val 340 345 350
Asp Arg Gly Val Glu Tyr Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys 70 355 360 365
- J> 66 Glu Lys He He Arg Thr Ser Asn Leu Asn Asp Ser Leu Gly Gin He 370 375 380
He Val Met Asp Ser He Gly Asn Asn Cys Thr Met Asn Phe Gin Asn 385 390 395 400
Asn Asn Gly Ser Asn He Gly Leu Leu Gly Phe His Ser Asn Asn Leu 405 410 415 Val Ala Ser Ser Trp Tyr Tyr Asn Asn He Arg Arg Asn Thr Ser Ser
420 425 430
Asn Gly Cys Phe Trp Ser Ser He Ser Lys Glu Asn Gly Trp Lys Glu 435 440 445
(2) INFORMATION FOR SEQ ID NO: 74:
U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear in) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
>;:ι; SEQUENCE DESCRIPTION: SEQ ID NO: 74:
CGCCATGGCT ATTCTAATTA TATATTTTAA TAG 33
(2) INFORMATION FOR SEQ ID NO: 75:
U) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
GCAAGCTTTC ATTCTTTCCA TCCATTCTC 29
> 2 ) INFORMATION FOR SEQ ID NO: 76:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3894 base pain (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
Ui) MOLECULE TYPE: DNA (genomic)
Ux ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..3891 (χι) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
ATG CCA GTT AAT ATA AAA AAC TTT AAT TAT AAT GAC CCT ATT AAT AAT 48
Met Pro Val Asn He Lys Asn Phe Asn Tyr Asn Asp Pro He Asn Asn 1 5 10 15
GAT GAC ATT ATT ATG ATG GAA CCA TTC AAT GAC CCA GGG CCA GGA ACA 96
Asp Asp He He Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30 TAT TAT AAA GCT TTT AGG ATT ATA GAT CGT ATT TGG ATA GTA CCA GAA 144 Tyr Tyr Lys Ala Phe Arg He He Asp Arg He Trp He Val Pro Glu 35 40 45
AGG TTT ACT TAT GGA TTT CAA CCT GAC CAA TTT AAT GCC AGT ACA GGA 192 Arg Phe Thr Tyr Gly Phe Gin Pro Asp Gin Phe Asn Ala Ser Thr Gly 50 55 60
GTT TTT AGT AAA GAT GTC TAC GAA TAT TAC GAT CCA ACT TAT TTA AAA 240 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80
ACC GAT GCT GAA AAA GAT AAA TTT TTA AAA ACA ATG ATT AAA TJA TTT 288 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met He Lys Leu Phe 85 90 ' 95
AAT AGA ATT AAT TCA AAA CCA TCA GGA CAG AGA TTA CTG GAT ATG ATA 336 Asn Arg He Asn Ser Lys Pro Ser Gly Gin Arg Leu Leu Asp Met He 100 105 110 GTA GAT GCT ATA CCT TAT CTT GGA AAT GCA TCT ACA CCG CCC GAC AAA 384 Val Asp Ala He Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys
115 120 125
TTT GCA GCA AAT GTT GCA AAT GTA TCT ATT AAT AAA AAA ATT ATC CAA 432 Phe Ala Ala Asn Val Ala Asn Val Ser He Asn Lys Lys He He Gin 130 135 140
CCT GGA GCT GAA GAT CAA ATA AAA GGT TTA ATG ACA AAT TTA ATA ATA 480 Pro Gly Ala Glu Asp Gin He Lys Gly Leu Met Thr Asn Leu He He 145 150 155 160
TTT GGA CCA GGA CCA GTT CTA AGT GAT AAT TTT ACT GAT AGT ATG ATT 528
Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met He
165 170 175
ATG AAT GGC CAT TCC CCA ATA TCA GAA GGA TTT GGT GCA AGA ATG ATG 576
Met Asn Gly His Ser Pro He Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190 ATA AGA TTT TGT CCT AGT TGT TTA AAT GTA TTT AAT AAT GTT CAG GAA 624 He Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gin Glu 195 200 205
AAT AAA GAT ACA TCT ATA TTT AGT AGA CGC GCG TAT TTT GCA GAT CCA 672 Aεn Lys Asp Thr Ser He Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220
GCT CTA ACG TTA ATG CAT GAA CTT ATA CAT GTG TTA CAT GGA TTA TAT 720 Ala Leu Thr Leu Met Hiε Glu Leu He His Val Leu Hiε Gly Leu Tyr 225 230 235 240
GGA ATT AAG ATA AGT AAT TTA CCA ATT ACT CCA AAT ACA AAA GAA TTT 768
Gly He Lys He Ser Asn Leu Pro He Thr Pro Asn Thr Lys Glu Phe
245 250 255
TTC ATG CAA CAT AGC GAT CCT GTA CAA GCA GAA GAA CTA TAT ACA TTC 816
Phe Met Gin His Ser Asp Pro Val Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GGA GGA CAT GAT CCT AGT GTT ATA AGT CCT TCT ACG GAT ATG AAT ATT 864 Gly Gly His Asp Pro Ser Val He Ser Pro Ser Thr Asp Met Asn He 275 280 285
TAT AAT AAA GCG TTA CAA AAT TTT CAA GAT ATA GCT AAT AGG CTT AAT 912 Tyr Asn Lys Ala Leu Gin Asn Phe Gin Asp He Ala Asn Arg Leu Asn 290 295 300
ATT GTT TCA AGT GCC CAA GGG AGT GGA ATT GAT ATT TCC TTA TAT AAA 960 He Val Ser Ser Ala Gin Gly Ser Gly He Asp He Ser Leu Tyr Lys 305 310 315 320 CAA ATA TAT AAA AAT AAA TAT GAT TTT GTT GAA GAT CCT AAT GGA AAA 1008
Gin He Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335
TAT AGT GTA GAT AAG GAT AAG TTT GAT AAA TTA TAT AAG GCC TTA ATG 1056
Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350
TTT GGC TTT ACT GAA ACT AAT CTA GCT GGT GAA TAT GGA ATA AAA ACT 1104
Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly He Lys Thr
355 360 365
AGG TAT TCT TAT TTT AGT GAA TAT TTG CCA CCG ATA AAA ACT GAA AAA 1152
Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro He Lys Thr Glu Lys 370 375 380
TTG TTA GAC AAT ACA ATT TAT ACT CAA AAT GAA GGC TTT AAC ATA GCT 1200
Leu Leu Asp Asn Thr He Tyr Thr Gin Asn Glu Gly Phe Asn He Ala 385 390 395 400
AGT AAA AAT CTC AAA ACG GAA TTT AAT GGT CAG AAT AAG GCG GTA AAT 1248
Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gin Asn Lys Ala Val Asn 405 410 415 AAA GAG GCT TAT GAA GAA ATC AGC CTA GAA CAT CTC GTT ATA TAT AGA 1296
Lyε Glu Ala Tyr Glu Glu He Ser Leu Glu His Leu Val He Tyr Arg 420 425 430
ATA GCA ATG TGC AAG CCT GTA ATG TAC AAA AAT ACC GGT AAA TCT GAA 1344 He Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu
435 440 445
CAG TGT ATT ATT GTT AAT AAT GAG GAT TTA TTT TTC ATA GCT AAT AAA 1392
Gin Cys He He Val Asn Asn Glu Asp Leu Phe Phe He Ala Asn Lys 450 455 460
GAT AGT TTT TCA AAA GAT TTA GCT AAA GCA GAA ACT ATA GCA TAT AAT 1440
Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr He Ala Tyr Asn 465 470 475 480
ACA CAA AAT AAT ACT ATA GAA AAT AAT TTT TCT ATA GAT CAG TTG ATT 1488
Thr Gin Asn Aεn Thr He Glu Asn Asn Phe Ser He Asp Gin Leu He 485 490 495 TTA GAT AAT GAT TTA AGC AGT GGC ATA GAC TTA CCA AAT GAA AAC ACA 1536
Leu Asp Asn Asp Leu Ser Ser Gly He Asp Leu Pro Asn Glu Asn Thr 500 505 510
GAA CCA TTT ACA AAT TTT GAC GAC ATA GAT ATC CCT GTG TAT ATT AAA 1584 Glu Pro Phe Thr Asn Phe Asp Asp He Asp He Pro Val Tyr He Lys
515 520 525
CAA TCT GCT TTA AAA AAA ATT TTT GTG GAT GGA GAT AGC CTT TTT GAA 1632
Gin Ser Ala Leu Lys Lys He Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540
TAT TTA CAT GCT CAA ACA TTT CCT TCT AAT ATA GAA AAT CTA CAA CTA 1680
Tyr Leu His Ala Gin Thr Phe Pro Ser Asn He Glu Asn Leu Gin Leu 545 550 555 560
ACG AAT TCA TTA AAT GAT GCT TTA AGA AAT AAT AAT AAA GTC TAT ACT 1728
Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575 TTT TTT TCT ACA AAC CTT GTT GAA AAA GCT AAT ACA GTT GTA GGT GCT 1776
Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590
TCA CTT TTT GTA AAC TGG GTA AAA GGA GTA ATA GAT GAT TTT ACA TCT 1824 Ser Leu Phe Val Asn Trp Val Lys Gly Val He Asp Asp Phe Thr Ser
569 595 600 605
GAA TCC ACA CAA AAA AGT ACT ATA GAT AAA GTT TCA GAT GTA TCC ATA 1872 Glu Ser Thr Gin Lys Ser Thr He Asp Lys Val Ser Asp Val Ser He 610 615 620
ATT ATT CCC TAT ATA GGA CCT GCT TTG AAT GTA GGA AAT GAA ACA GCT 1920 He He Pro Tyr He Gly Pro Ala Leu Asn Val Gly Aεn Glu Thr Ala 625 630 635 640
AAA GAA AAT TTT AAA AAT GCT TTT GAA ATA GGT GGA GCC GCT ATC TTA 1968 Lys Glu Asn Phe Lys Asn Ala Phe Glu He Gly Gly Ala Ala lie Leu 645 650 655 ATG GAG TTT ATT CCA GAA CTT ATT GTA CCT ATA GTT GGA TTT TTT ACA 2016 Met Glu Phe He Pro Glu Leu He Val Pro He Val Gly Phe Phe Thr 660 665 670
TTA GAA TCA TAT GTA GGA AAT AAA GGG CAT ATT ATT ATG ACG ATA TCC 2064 Leu Glu Ser Tyr Val Gly Asn Lys Gly His He He Met Thr He Ser 675 680 685
AAT GCT TTA AAG AAA AGG GAT CAA AAA TGG ACA GAT ATG TAT GGT TTG 2112 Asn Ala Leu Lys Lys Arg Asp Gin Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700
ATA GTA TCG CAG TGG CTC TCA ACG GTT AAT ACT CAA TTT TAT ACA ATA 2160 He Val Ser Gin Trp Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr He 705 710 715 720
AAA GAA AGA ATG TAC AAT GCT TTA AAT AAT CAA TCA CAA GCA ATA GAA 2208 Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gin er Gin Ala He Glu 725 730 735 AAA ATA ATA GAA GAT CAA TAT AAT AGA TAT AGT GAA GAA GAT AAA ATG 2256 Lys He He Glu Asp Gin Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 50
AAT ATT AAC ATT GAT TTT AAT GAT ATA GAT TTT AAA CTT AAT CAA AGT 2304 Asn He Aεn He Asp Phe Asn Asp He Asp Phe Lys Leu Asn Gin Ser 755 760 765
ATA AAT TTA GCA ATA AAC AAT ATA GAT GAT TTT ATA AAC CAA TGT TCT 2352 He Asn Leu Ala He Asn Asn He Asp Asp Phe He Asn Gin Cys Ser 770 775 780
ATA TCA TAT CTA ATG AAT AGA ATG ATT CCA TTA GCT GTA AAA AAG TTA 2400
He Ser Tyr Leu Met Asn Arg Met He Pro Leu Ala Val Lys Lys Leu 785 790 795 800
AAA GAC TTT GAT GAT AAT CTT AAG AGA GAT TTA TTG GAG TAT ATA GAT 2448
Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr He Asp 805 810 815 ACA AAT GAA CTA TAT TTA CTT GAT GAA GTA AAT ATT CTA AAA TCA AAA 2496 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn He Leu Lys Ser Lys 820 825 830
GTA AAT AGA CAC CTA AAA GAC AGT ATA CCA TTT GAT CTT TCA CTA TAT 2544 Val Asn Arg His Leu Lys Asp Ser He Pro Phe Asp Leu Ser Leu Tyr 835 840 845
ACC AAG GAC ACA ATT TTA ATA CAA GTT TTT AAT AAT TAT ATT AGT AAT 2592 Thr Lys Asp Thr He Leu He Gin Val Phe Asn Asn Tyr He Ser Asn 850 855 860
ATT AGT AGT AAT GCT ATT TTA AGT TTA AGT TAT AGA GGT GGG CGT TTA 2640 He Ser Ser Asn Ala He Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880 ATA GAT TCA TCT GGA TAT GGT GCA ACT ATG AAT GTA GGT TCA GAT GTT 2688
He Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 690 895 ATC TTT AAT GAT ATA GGA AAT GGT CAA TTT AAA TTA AAT AAT TCT GAA 2736
He Phe Asn Asp He Gly Asn Gly Gin Phe Lys Leu Aεn Asn Ser Glu 900 905 910
AAT AGT AAT ATT ACG GCA CAT CAA AGT AAA TTC GTT GTA TAT GAT AGT 2784 Asn Ser Asn He Thr Ala His Gin Ser Lys Phe Val Val Tyr Asp Ser 915 920 925
ATG TTT GAT AAT TTT AGC ATT AAC TTT TGG GTA AGG ACT CCT AAA TAT 2832
Met Phe Asp Asn Phe Ser He Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940
AAT AAT AAT GAT ATA CAA ACT TAT CTT CAA AAT GAG TAT ACA ATA ATT 2880
Asn Asn Asn Asp He Gin Thr Tyr Leu Gin Asn Glu Tyr Thr He He 945 950 955 960
AGT TGT ATA AAA AAT GAC TCA GGA TGG AAA GTA TCT ATT AAG GGA AAT 2928
Ser Cys He Lys Asn Asp Ser Gly Trp Lys Val Ser He Lyε Gly Asn 965 970 975 AGA ATA ATA TGG ACA TTA ATA GAT GTT AAT GCA AAA TCT AAA TCA ATA 2976
Arg He He Trp Thr Leu He Asp Val Asn Ala Lyε Ser Lys Ser He 980 985 990
TTT TTC GAA TAT AGT ATA AAA GAT AAT ATA TCA GAT TAT ATA AAT AAA 3024 Phe Phe Glu Tyr Ser He Lys Asp Aεn He Ser Asp Tyr He Asn Lys 995 1000 1005
TGG TTT TCC ATA ACT ATT ACT AAT GAT AGA TTA GGT AAC GCA AAT ATT 3072
Trp Phe Ser He Thr He Thr Asn Asp Arg Leu Gly Asn Ala Asn He 1010 1015 1020
TAT ATA AAT GGA AGT TTG AAA AAA AGT GAA AAA ATT TTA AAC TTA GAT 3120
Tyr He Asn Gly Ser Leu Lys Lys Ser Glu Lys He Leu Asn Leu Asp 1025 1030 1035 1040
AGA ATT AAT TCT AGT AAT GAT ATA GAC TTC AAA TTA ATT AAT TGT ACA 3168
Arg He Asn Ser Ser Asn Asp He Asp Phe Lys Leu He Asn Cys Thr 1045 1050 1055 GAT ACT ACT AAA TTT GTT TGG ATT AAG GAT TTT AAT ATT TTT GGT AGA 3216
Asp Thr Thr Lys Phe Val Trp He Lys Asp Phe Asn He Phe Gly Arg 1060 1065 1070
GAA TTA AAT GCT ACA GAA GTA TCT TCA CTA TAT TGG ATT CAA TCA TCT 3264 Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr Trp He Gin Ser Ser 1075 1080 1085
ACA AAT ACT TTA AAA GAT TTT TGG GGG AAT CCT TTA AGA TAC GAT ACA 3312
Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr 1090 1095 1100
CAA TAC TAT CTG TTT AAT CAA GGT ATG CAA AAT ATC TAT ATA AAG TAT 3360
Gin Tyr Tyr Leu Phe Asn Gin Gly Met Gin Asn He Tyr He Lys Tyr 1105 ' 1110 1115 ' 1120
TTT AGT AAA GCT TCT ATG GGG GAA ACT GCA CCA CGT ACA AAC TTT AAT 3408 Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Asn 1125 1130 1135 AAT GCA GCA ATA AAT TAT CAA AAT TTA TAT CTT GGT TTA CGA TTT ATT 3456 Asn Ala Ala He Asn Tyr Gin Asn Leu Tyr Leu Gly Leu Arg Phe He 1140 1145 1150
ATA AAA AAA GCA TCA AAT TCT CGG AAT ATA AAT AAT GAT AAT ATA GTC 3504 He Lys Lys Ala Ser Asn Ser Arg Asn He Asn Asn Asp Asn He Val 1155 1160 1165
AGA GAA GGA GAT TAT ATA TAT CTT AAT ATT GAT AAT ATT TCT GAT GAA 3552 Arg Glu Gly Asp Tyr He Tyr Leu Asn He Asp Aεn He Ser Asp Glu 1170 1175 1180
TCT TAC AGA GTA TAT GTT TTG GTG AAT TCT AAA GAA ATT CAA ACT CAA 3600 Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu He Gin Thr Gin 1185 1190 1195 1200
TTA TTT TTA GCA CCC ATA AAT GAT GAT CCT ACG TTC TAT GAT GTA CTA 3648 Leu Phe Leu Ala Pro He Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215
CAA ATA AAA AAA TAT TAT GAA AAA ACA ACA TAT AAT TGT CAG ATA CTT 3696 Gin He Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gin He Leu 1220 1225 1230 TGC GAA AAA GAT ACT AAA ACA TTT GGG CTG TTT GGA ATT GGT AAA TTT 3744 Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly He Gly Lys Phe 1235 1240 1245
GTT AAA GAT TAT GGA TAT GTT TGG GAT ACC TAT GAT AAT TAT TTT TGC 3792 Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250 1255 1260
ATA AGT CAG TGG TAT CTC AGA AGA ATA TCT GAA AAT ATA AAT AAA TTA 3840 He Ser Gin Trp Tyr Leu Arg Arg He Ser Glu Asn He Asn Lys Leu 1265 1270 1275 ' 1280
AGG TTG GGA TGT AAT TGG CAA TTC ATT CCC GTG GAT GAA GGA TGG ACA 3888 Arg Leu Gly Cys Asn Trp Gin Phe He Pro Val Asp Glu Gly Trp Thr 1285 1290 1295
GAA TAA 3894
Glu
(2) INFORMATION FOR SEQ ID NO: 77:
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1297 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protem
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: Met Pro Val Asn He Lys Asn Phe Asn Tyr Asn Asp Pro He Asn Asn 1 5 ' 10 15
Asp Asp He He Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30
Tyr Tyr Lys Ala Phe Arg He He Asp Arg He Trp He Val Pro Glu 35 40 45
Arg Phe Thr Tyr Gly Phe Gin Pro Asp Gin Phe Asn Ala Ser Thr Gly 50 55 60
Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met He Lys Leu Phe 85 90 95
Asn Arg He Asn Ser Lys Pro Ser Gly Gin Arg Leu Leu Asp Met He 100 105 110
Val Asp Ala He Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys
115 ' 120 125
Phe Ala Ala Asn Val Ala Asn Val Ser He Asn Lys Lys He He Gin 130 135 140
Pro Gly Ala Glu Asp Gin He Lys Gly Leu Met Thr Asn Leu He He 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser Aεp Asn Phe Thr Asp Ser Met He
165 170 175
Met Asn Gly His Ser Pro He Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190
He Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gin Glu 195 200 205
Asn Lys Asp Thr Ser He Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220
Ala Leu Thr Leu Met His Glu Leu He His Val Leu His Gly Leu Tyr 225 230 235 240 Gly He Lys He Ser Asn Leu Pro He Thr Pro Asn Thr Lys Glu Phe
245 250 255
Phe Met Gin His Ser Asp Pro Val Gin Ala Glu Glu Leu Tyr Thr Phe 260 265 270
Gly Gly His Asp Pro Ser Val He Ser Pro Ser Thr Asp Met Asn He 275 280 285
Tyr Asn Lys Ala Leu Gin Aεn Phe Gin Asp He Ala Asn Arg Leu Asn 290 295 300
He Val Ser Ser Ala Gin Gly Ser Gly He Asp He Ser Leu Tyr Lys
305 310 315 .320 Gin He Tyr Lvs Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys
325 330 335
Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 ' 345 350
Phe Gly Phe Thr Glu Thr Aεn Leu Ala Gly Glu Tyr Gly He Lyε Thr 355 360 365
Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro He Lys Thr Glu Lys 370 375 380
Leu Leu Asp Asn Thr He Tyr Thr Gin Asn Glu Gly Phe Asn He Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gin Asn Lys Ala Val Asn
405 410 415
Lys Glu Ala Tyr Glu Glu He Ser Leu Glu His Leu Val He Tyr Arg
420 425 430
He Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu
435 ' 440 445
Gin Cys He He Val Asn Asn Glu Asp Leu Phe Phe He Ala Asn Lyε 450 455 460 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr He Ala Tyr Asn 465 470 475 480
Thr Gin Asn Asn Thr He Glu Asn Asn Phe Ser He Asp Gin Leu He 485 490 495
Leu Asp Asn Asp Leu Ser Ser Gly He Asp Leu Pro Asn Glu Asn Thr 500 505 510
Glu Pro Phe Thr Asn Phe Asp Asp He Asp He Pro Val Tyr He Lys 515 520 525
Gin Ser Ala Leu Lys Lys He Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540
Tyr Leu His Ala Gin Thr Phe Pro Ser Asn He Glu Asn Leu Gin Leu 545 550 555 560
Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Aεn Asn Lys Val Tyr Thr 565 570 575
Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590
Ser Leu Phe Val Asn Trp Val Lys Gly Val He Asp Asp Phe Thr Ser 595 600 605
Glu Ser Thr Gin Lys Ser Thr He Asp Lys Val Ser Asp Val Ser He 610 615 620
He He Pro Tyr He Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala 625 630 635 640
Lys Glu Asn Phe Lys Asn Ala Phe Glu He Gly Gly Ala Ala He Leu 645 650 655
Met Glu Phe He Pro Glu Leu He Val Pro He Val Gly Phe Phe Thr 660 665 670 Leu Glu Ser Tyr Val Gly Asn Lys Gly His He He Met Thr He Ser 675 680 685
Asn Ala Leu Lys Lys Arg Asp Gin Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700
He Val Ser Gin Trp Leu Ser Thr Val Asn Thr Gin Phe Tyr Thr He 705 710 715 720
Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gin Ser Gin Ala He Glu 725 730 735
Lys He He Glu Asp Gin Tyr Asn Arg Tyr Ser Glu Glu Asp Lyε Met
740 745 750 Asn He Asn He Asp Phe Asn Asp He Asp Phe Lys Leu Asn Gin Ser
755 760 765
He Asn Leu Ala He Asn Asn He Asp Asp Phe He Asn Gin Cys Ser
770 775 780
He Ser Tyr Leu Met Asn Arg Met He Pro Leu Ala Val Lyε Lyε Leu
785 ' 790 795 800
Lyε Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr He Asp 805 810 815
Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn He Leu Lys Ser Lys
820 825 830 Val Asn Arg His Leu Lys Asp Ser He Pro Phe Asp Leu Ser Leu Tyr 835 840 845
Thr Lys Asp Thr He Leu He Gin Val Phe Asn Asn Tyr He Ser Asn 850 855 860
5
He Ser Ser Asn Ala He Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880
He Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 10 885 890 895
He Phe Asn Asp He Gly Asn Gly Gin Phe Lys Leu Asn Asn Ser Glu 900 905 910
15 Asn Ser Asn He Thr Ala His Gin Ser Lys Phe Val Val Tyr Asp Ser 915 920 925
Met Phe Asp Asn Phe Ser He Asn Phe Trp Val Arg Thr Pro Lys Tyr
930 935 940
20
Asn Asn Aεn Asp He Gin Thr Tyr Leu Gin Aεn Glu Tyr Thr He He 945 950 955 960
Ser Cys He Lys Asn Asp Ser Gly Trp Lys Val Ser He Lyε Gly Asn 965 970 975
Λzg He He Trp Thr Leu He Asp Val Aεn Ala Lyε Ser Lyε Ser He 980 985 ' 990
30 Phe Phe Glu Tyr Ser He Lys Asp Asn He Ser Aεp Tyr He Asn Lyε 995 ' 1000 1005
Trp Phe Ser He Thr He Thr Asn Asp Arg Leu Gly Asn Ala Asn He 1010 1015 * 1020
.1.1
Tyr He Asn Gly Ser Leu Lys Lys Ser Glu Lys He Leu Asn Leu Aεp
102 1030 1035 1040
Arg He Asn Ser Ser Asn Asp He Asp Phe Lys Leu He Asn Cyε Thr 40 1045 1050 1055
Asp Thr Thr Lys Phe Val Trp He Lys Asp Phe Asn He Phe Gly Arg 1060 1065 1070
4. >,iu L°u Aεn Ara Thr Glu Val Ser Ser Leu Tyr Trp He Gin Ser Ser
1075 1080 1085
Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Aεp Thr
1090 1095 ' 1100
50
Gin Tyr Tyr Leu Phe Asn Gin Gly Met Gin Asn He Tyr He Lys Tyr
1105 1110 1115 1120
Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro Arg Thr Asn Phe Aεn
55 1125 1130 1135
Asn Ala Ala He Asn Tyr Gin Asn Leu Tyr Leu Gly Leu Arg Phe He
1140 1145 1150
60 He Lys Lys Ala Ser Asn Ser Arg Asn He Asn Asn Asp Asn He Val
1155 1160 1165
Arg Glu Gly Asp Tyr He Tyr Leu Asn He Asp Asn He Ser Asp Glu 1170 1175 1180
65
Ser Tyr Arg Val Tyr Val Leu Val Aεn Ser Lys Glu He Gin Thr Gin 1185 * 1190 1195 1200
Leu Phe Leu Ala Pro He Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu 70 1205 1210 1215 Gin He Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gin He Leu 1220 1225 1230
Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly He Gly Lys Phe 1235 1240 1245
Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys 1250 1255 1260 He Ser Gin Trp Tyr Leu Arg Arg He Ser Glu Asn He Asn Lys Leu J265 1270 1275 1280
Arg Leu Gly Cys Asn Trp Gin Phe He Pro Val Asp Glu Gly Trp Thr 1285 1290 1295
Glu
(2) INFORMATION FOR SEQ ID NO: 78: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1535 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear ii) MOLECULE TYPE: DNA (genomic) ix) FEATURE:
(A) NAME/KEY: CDS (B) LOCATION: 108..1526
(κi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
AGATCTCGAT CCCGCGAAAT TAATACGACT CACTATAGGG GAATTGTGAG CGGATAACAA 60
TTCCCCTCTA GAAATAATTT TGTTTAACTT TAAGAAGGAG ATATACC ATG GGC CAT 116
Met Gly His
1 CAT CAT CAT CAT CAT CAT CAT CAT CAC AGC AGC GGC CAT ATC GAA GGT 164 His His His His His His His His His Ser Ser Gly His He Glu Gly 5 10 15
CGT CAT ATG GCT AGC ATG GCT GAC ACA ATT TTA ATA CAA GTT TTT AAT 212 Arg His Met Ala Ser Met Ala Asp Thr He Leu He Gin Val Phe Asn 20 25 30 35
AAT TAT ATT AGT AAT ATT AGT AGT AAT GCT ATT TTA AGT TTA AGT TAT 260 Asn Tyr He Ser Asn He Ser Ser Asn Ala He Leu Ser Leu Ser Tyr 40 45 50
AGA GGT GGG CGT TTA ATA GAT TCA TCT GGA TAT GGT GCA ACT ATG AAT 308 Arg Gly Gly Arg Leu He Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn 55 60 65
GTA GGT TCA GAT GTT ATC TTT AAT GAT ATA GGA AAT GGT CAA TTT AAA 356 Val Gly Ser Asp Val He Phe Asn Asp He Gly Asn Gly Gin Phe Lys 70 75 80 TTA AAT AAT TCT GAA AAT AGT AAT ATT ACG GCA CAT CAA AGT AAA TTC 404 Leu Asn Asn Ser Glu Asn Ser Asn He Thr Ala His Gin Ser Lys Phe 85 90 95
GTT GTA TAT GAT AGT ATG TTT GAT AAT TTT AGC ATT AAC TTT TGG GTA 452 Val Val Tyr Asp Ser Met Phe Asp Asn Phe Ser He Asn Phe Trp Val 100 105 110 115
AGG ACT CCT AAA TAT AAT AAT AAT GAT ATA CAA ACT TAT CTT CAA AAT 500 Arg Thr Pro Lys Tyr Asn Asn Aεn Aεp He Gin Thr Tyr Leu Gin Asn ' 120 125 ' 130 GAG TAT ACA ATA ATT AGT TGT ATA AAA AAT GAC TCA GGA TGG AAA GTA 548 Glu Tyr Thr He He Ser Cys He Lys Asn Asp Ser Gly Trp Lys Val 135 140 145 TCT ATT AAG GGA AAT AGA ATA ATA TGG ACA TTA ATA GAT GTT AAT GCA 596 Ser He Lys Gly Asn Arg He He Trp Thr Leu He Asp Val Asn Ala 150 155 160
AAA TCT AAA TCA ATA TTT TTC GAA TAT AGT ATA AAA GAT AAT ATA TCA 644 Lys Ser Lys Ser He Phe Phe Glu Tyr Ser He Lys Asp Asn He Ser 165 ' 170 175
GAT TAT ATA AAT AAA TGG TTT TCC ATA ACT ATT ACT AAT GAT AGA TTA 692 Aεp Tyr He Asn Lys Trp Phe Ser He Thr He Thr Asn Asp Arg Leu 180 ' 185 190 195
GGT AAC GCA AAT ATT TAT ATA AAT GGA AGT TTG AAA AAA AGT GAA AAA 740
Gly Asn Ala Asn He Tyr He Asn Gly Ser Leu Lys Lys Ser Glu Lys 200 ' 205 210
ATT TTA AAC TTA GAT AGA ATT AAT TCT AGT AAT GAT ATA GAC TTC AAA 788
He Leu Asn Leu Asp Arg He Asn Ser Ser Aεn Asp He Asp Phe Lyε 215 220 225 TTA ATT AAT TGT ACA GAT ACT ACT AAA TTT GTT TGG ATT AAG GAT TTT 836 Leu He Aεn Cys Thr Asp Thr Thr Lys Phe Val Trp He Lys Asp Phe 230 ' 235 240
AAT ATT TTT GGT AGA GAA TTA AAT GCT ACA GAA GTA TCT TCA CTA TAT 884 Asn He Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr 245 250 255
TGG ATT CAA TCA TCT ACA AAT ACT TTA AAA GAT TTT TGG GGG AAT CCT 932 Trp He Gin Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro 260 265 270 ' 275
TTA AGA TAC GAT ACA CAA TAC TAT CTG TTT AAT CAA GGT ATG CAA AAT 980
Leu Arg Tyr Asp Thr Gin Tyr Tyr Leu Phe Asn Gin Gly Met Gin Asn
280 285 290
ATC TAT ATA AAG TAT TTT AGT AAA GCT TCT ATG GGG GAA ACT GCA CCA 1028
He Tyr He Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu Thr Ala Pro
295 300 305 CGT ACA AAC TTT AAT AAT GCA GCA ATA AAT TAT CAA AAT TTA TAT CTT 1076 Arg Thr Asn Phe Asn Asn Ala Ala He Asn Tyr Gin Asn Leu Tyr Leu 310 315 320
GGT TTA CGA TTT ATT ATA AAA AAA GCA TCA AAT TCT CGG AAT ATA AAT 1124 Gly Leu Arg Phe He He Lys Lys Ala Ser Asn Ser Arg Asn He Asn 325 330 ' 335
AAT GAT AAT ATA GTC AGA GAA GGA GAT TAT ATA TAT CTT AAT ATT GAT 1172 Asn Asp Asn He Val Arg Glu Gly Asp Tyr He Tyr Leu Asn He Asp 340 345 350 355
AAT ATT TCT GAT GAA TCT TAC AGA GTA TAT GTT TTG GTG AAT TCT AAA 1220
Asn He Ser Asp Glu Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys
360 365 370
GAA ATT CAA ACT CAA TTA TTT TTA GCA CCC ATA AAT GAT GAT CCT ACG 1268
Glu He Gin Thr Gin Leu Phe Leu Ala Pro He Asn Asp Asp Pro Thr 375 380 385 TTC TAT GAT GTA CTA CAA ATA AAA AAA TAT TAT GAA AAA ACA ACA TAT 1316 Phe Tyr Asp Val Leu Gin He Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr 390 395 400
AAT TGT CAG ATA CTT TGC GAA AAA GAT ACT AAA ACA TTT GGG CTG TTT 1364 Asn Cys Gin He Leu Cys Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe 405 410 415
GGA ATT GGT AAA TTT GTT AAA GAT TAT GGA TAT GTT TGG GAT ACC TAT 1412 Gly He Gly Lys Phe Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr 420 425 430 435
GAT AAT TAT TTT TGC ATA AGT CAG TGG TAT CTC AGA AGA ATA TCT GAA 1460 Asp Asn Tyr Phe Cys He Ser Gin Trp Tyr Leu Arg Arg He Ser Glu 440 445 450
AAT ATA AAT AAA TTA AGG TTG GGA TGT AAT TGG CAA TTC ATT CCC GTG 1508 Asn He Asn Lys Leu Arg Leu Gly Cys Asn Trp Gin Phe He P_ro Val 455 460 465
GAT GAA GGA TGG ACA GAA TAACTCGAG 1535
Asp Glu Gly Trp Thr Glu 470
(2) INFORMATION FOR SEQ ID NO: 79:
H) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 473 ammo acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
Ui) MOLECULE TYPE: protem
(κi) SEQUENCE DESCRIPTION. SEQ ID NO: 79. Met Gly His His His His His His His His His His Ser Ser Gly His
1 5 10 15
He Glu Gly Arg His Met Ala Ser Met Ala Asp Thr He Leu He Gin 20 25 30
Val Phe Asn Asn Tyr He Ser Aεn He Ser Ser Asn Ala He Leu Ser 35 40 45
Leu Ser Tyr Arg Gly Gly Arg Leu He Asp Ser Ser Gly Tyr Gly Ala 50 55 60
Thr Met Asn Val Gly Ser Aεp Val He Phe Asn Asp He Gly Asn Gly
65 ' 70 75 80 Gin Phe Lys Leu Asn Asn Ser Glu Asn Ser Asn He Thr Ala His Gin
85 90 95
Ser Lys Phe Val Val Tyr Asp Ser Met Phe Asp Asn Phe Ser He Asn 100 105 110
Phe Trp Val Arg Thr Pro Lys Tyr Asn Asn Asn Asp He Gin Thr Tyr 115 120 125
Leu Gin Asn Glu Tyr Thr He He Ser Cys He Lys Asn Aεp Ser Gly 130 135 140
Trp Lys Val Ser He Lys Gly Asn Arg He He Trp Thr Leu He Asp
145 150 ' 155 160 Val Asn Ala Lys Ser Lys Ser He Phe Phe Glu Tyr Ser He Lys Asp
165 170 175
Asn He Ser Asp Tyr He Asn Lys Trp Phe Ser He Thr He Thr Asn 180 185 190
Asp Arg Leu Gly Asn Ala Asn He Tyr He Asn Gly Ser Leu Lys Lys 195 " 200 205
Ser Glu Lys He Leu Asn Leu Asp Arg He Asn Ser Ser Asn Asp He 210 215 220 Asp Phe Lys Leu He Asn Cys Thr Asp Thr Thr Lys Phe Val Trp He 225 230 235 240
Lys Asp Phe Asn He Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser 245 250 255
Ser Leu Tyr Trp He Gin Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp 260 265 270
Gly Asn Pro Leu Arg Tyr Asp Thr Gin Tyr Tyr Leu Phe Asn Gin Gly 275 280 285
Met Gin Aεn He Tyr He Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu 290 295 300
15
Thr Ala Pro Arg Thr Aεn Phe Asn Asn Ala Ala He Asn Tyr Gin Asn 305 310 315 320
Leu Tyr Leu Gly Leu Arg Phe He He Lys Lys Ala Ser Asn Ser Arg 325 330 335
Asn He Asn Asn Asp Asn He Val Arg Glu Gly Asp Tyr He Tyr Leu 340 345 350
">i Asn He Asp Asn He Ser Asp Glu Ser Tyr Arg Val Tyr Val Leu Val 355 360 ' 365
Asn Ser Lys Glu He Gin Thr Gin Leu Phe Leu Ala Pro He Asn Asp 370 375 380
30
Asp Pro Thr Phe Tyr Asp Val Leu Gin He Lys Lys Tyr Tyr Glu Lvs 385 390 395 ' 400
Thr Thr Tyr Asn Cys Gin He Leu Cys Glu Lys Asp Thr Lys Thr Phe
55 405 410 415
Gly Leu Phe Gly He Gly Lyε Phe Val Lys Asp Tyr Gly Tyr Val Trp 420 425 430
40 Asp Thr Tyr Asp Asn Tyr Phe Cys He Ser Gin Trp Tyr Leu Arg Arg 435 440 445
He Ser Glu Asn He Asn Lys Leu Arg Leu Gly Cys Asn Trp Gin Phe
450 455 460
45
He Pro Val Asp Glu Gly Trp Thr Glu 465 470
(2) INFORMATION FOR SEQ ID NO: 80:
50
U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single 55 (D) TOPOLOGY: linear
Ui) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
60 (κi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
CGCCATGGCT GACACAATTT TAATACAAGT 30
(2) INFORMATION FOR SEQ ID NO: 81: 65
( l ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single 70 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA"
(Xl) SEQUENCE DESCRIPTION: SEQ ID N0:81:
GCCTCGAGTT ATTCTGTCCA TCCTTCATCC AC (2) INFORMATION FOR SEQ ID NO: 62: U) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 ammo acids
Figure imgf000382_0001
(C) STRANDEDNESS: not relevant
(D) TOPOLOGY: not relevant
Ui) MOLECULE TYPE: peptide iix) FEATURE:
(A) NAME/KEY: Modifled-site (B) LOCATION: 12
(D) OTHER INFORMATION: /note= "The asparagine residue at this position contains an amide group."
(κi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
Cys Gin Thr He Asp Gly Lys Lys Tyr Tyr Phe Asn 1* 5 10

Claims

1 A host cell containing a recombinant expression vector, said vector encoding a protein comprising at least a portion of a Clostridium botulinum toxin, said toxm selected Irom the group consisting of type B toxin and type E toxin
2 T he host cell ol Claim 1. wherein and said host cell is capable ol expressing said protein at a level greater than or equal to 5% of the total cellular protein
3 The host cell of Claim 1 , wherein and said host cell is capable of expressing said protein as a soluble protein at a level greater than or equal to 0 25% ol the total soluble cellular protein
4 The host cell of Claim 1. wherein said host cell is an Escherichia co t ll
5 I he host cell of Claim 1. wherein said host cell is an insect cell
6 The host cell ol Claim 1. wherein said host cell is a yeast cell
7 A host cell containing a recombinant expression vector, said vector encoding a fusion protein comprising a non-toxin protein sequence and at least a portion ol a ( losli idium hoiulinum toxin, said toxin selected from the group consisting of type B toxin and tvpe f toxin
8 The host cell of Claim 7. wherein said portion of said toxm comprises the receptor binding domain
9 The host cell of Claim 7, wherein said non-toxin protein sequence comprises a poly-histidine tract
10 A vaccine comprising a fusion protein, said fusion protein comprising a non- toxin protein sequence and at least a portion of a Clostridium botulinum toxin, said toxin selected from the group consisting of type B toxin and type E toxin
1 1 The vaccine of Claim 10 further compπsing a fusion protein compπsing a non- toxin protein sequence and at least a portion of Clostridium botulinum type A toxin
12 The vaccine of Claim 1 . wherein said portion of said Clostridium botulinum toxin comprises the receptor binding domain
13 The vaccine of Claim 10 wherein said non-toxin protein sequence comprises a poly-histidine tract
14 The vaccine of Claim 10, wherein said vaccine is substantially endotoxin-free
15 A method of generating antibody directed against a Clostridium botulinum toxin comprising a) providing in any order i) an antigen compπsing a fusion protein comprising a non-toxin protein sequence and at least a portion of a Clostridium hoiulinum toxin, said toxin selected from the group consisting of type B toxin and type F toxin, and n) a host, and b) immunizing said host with said antigen so as to generate an antibody
16 The method of Claim 15, wherein said antigen further comprises a fusion protein comprising a non-toxin protein sequence and at least a portion ot Clostridium hoiulinum type A toxin
17 The method of Claim 15, wherein said portion ol said Clostridium botulinum toxin comprises the receptor binding domain
18 T he method of Claim 15 wherein said non-toxin protein sequence comprises a poly-histidine tract
19 The method of Claim 15 wherein said host is a mammal
20 T he method of Claim 19 wherein said mammal is a human
21. The method of Claim 15 further comprising step c) collecting said antibodies from said host.
22. The method of Claim 21 further comprising step d) purifying said antibodies.
23. The antibody raised according to the method of Claim 15.
24. The antibody raised according to the method of Claim 16.
583
PCT/US1997/015394 1996-08-28 1997-08-28 Multivalent vaccine for clostridium botulinum neurotoxin WO1998008540A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
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CA002296765A CA2296765A1 (en) 1996-08-28 1997-08-28 Soluble recombinant botulinum toxin proteins
AU42450/97A AU4245097A (en) 1996-08-28 1997-08-28 Multivalent vaccine for (clostridium botulinum) neurotoxin

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US08/704,159 1996-08-28
US08/704,159 US6967088B1 (en) 1995-03-16 1996-08-28 Soluble recombinant botulinum toxin proteins

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US7052702B1 (en) 1997-10-08 2006-05-30 Health Protection Agency Conjugates of galactose-binding lectins and clostridial neurotoxins as analgesics
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US7740868B2 (en) 1999-08-25 2010-06-22 Allergan, Inc. Activatable clostridial toxins
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