CA1265077A - Hybrid proteins produced by an ultrahigh prokaryotic expression system - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Hybrid useful proteins are prepared by a novel biological system comprising a prokaryotic host trans-formed with novel hybrid plasmids' .beta.-glucuronidase (BG) gene DNA and the desired protein gene DNA. Specifically exemplified are plasmids which comprise BG gene DNA and protein A DNA. E. coli K-12 derivative hosts transformed with plasmid pBG3-2.DELTA.N express >60% of the desired fusion protein having protein A-like biological activity.
Other useful proteins can be expressed via the elegant highly efficient expression system of the subject inven-tion.

Description

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DESCRIPTION

HYBRID PROTEINS PRODIJCED BY AN
ULTRAHIG~ PROKARYOTIC EXPRESSION SYSTEM

BACKG~OUND OF THE INVENTION

Expression level is one of the most important considerations in the utilization of cloned gene products. Elevated levels of protein expression have important ramifications both in terms of protein yield per fermentation volume and in degree of purification difficulty. Most efforts at increasing expression of cloned gene products have, to date, focused on the use of strong promoters in conjunction with an efficient ribosome binding site. A variety of promoters have been used to increase expression, the most commonly used being the PL ~romoter from phage lambda and the E. coli lacUV5 and trp promoters.
The lambda PL promoter has been successfully used in conjunction with a CI857 temperature-sensitive lambda repressor. This allows for low level expression of the cloned product during E. coli ~rowth at 3~C. Once substan~ial cell density is established, the cloned gene can be derepressed by growth at 42C. This method has been used in the expression of gene products lethal to the host cells. Several investigators have reported ~5 expression levels of 4% (Waldman, A.S., Haensslein, E., and Milman, G.[1983] J. Bio. Chem. 258:1157I-11575); 7%
(Yoakum, G.H., Yeung, A.T., Mattes, W.B., and Grossman, L. ~I982] PNAS ,9:1766-1770; Derom, C., Gheysen, D., and Fiers, W., ll982] Gene, 17:45-54); and 13% (Oehr-nichen, R., Klocl;, G., Alts~hmid, L., and Hillen, W.
[1984] EMBO J. 3:539-543) USlng the PL promoter under , ~ ~

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-2- R126 thermolabile repressor con~rol.
Recently, there has been increased use of a chimeric promoter consisting of sequences from the E. coli lacUV5 and trp promoters. This hybrid promoter is known as the tac promoter; it contains the -10 region from the lac promoter and the -35 region of tr~. This hybrid promoter is repressed by the E. coli lac Iq gene product and induced by 5 mM isopropyl-~-D-thio-nalactop~ranocide (IPTG). This system has been used by several investigators with varying results. Ex-pression of various proteins have reached the 7~ level (Bagdasarian, M.~l., Amann, E., Lurz, R., Ruckert, B., and Bogdasarian, M. [1983] Gene 26:273-282);the 10~
level (Bikel, I., Roberts, T.~l., Bladon, M.T., Green, R., Amann, E. and Livingston, D.M. [1983] PNAS 80:906-910) and the 30% level (Amann, E., Brosius, J., and Ptashne, M. [1983] Gene 25:167-178).
Protein expression levels are dependent on the genetic background of the host cell. The utilization ~0 of host cells containing specific mutations has been shown to increase the level of a cloned protein. Two genes have received wide attention in this regard, `the lon and ~ mutations.
The lon mutation has been map~ed to the capR
region o~ the E. coli genome and has been shown to code for an ATP-dependent protease (Bukhari, A.I.
and Zipser, D., [1973] J. Bact. 116:1469-1471; Shine-berg, B. and Zipser, D., [1973] J. Bact. 116:1469-1471). This ATP-dependent ~rotease is one of the eight proteases found in E. coli (Chung, C.H. and Goldberg, A.L. [1981] PNAS 78:4931-4935; Sreedhara Swamy, K.H.
and Goldberg, A.L. [1981] Nature 292:652-654). It has been demonstrated to be the major protease involved in the degradation of prot~lns produced from missense .
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-3- R126 and nonsense mutations (Mount, D.W. [1980~ Ann Rev.
Genet~ 14:297-319). The pnp mutation has been mapped to the polyribonucleotide phosphorylase gene. Polyribo-nucleotide phosphorylase has been shown to be involved in the phosphorolysis of ribonucleic acid and therefore implicated in mRNA breakdown. Subsequent studies have shown a 20- ~o 100-fold increase in specific activity of cloned fungal catabolite dehydrogenase when cloned into pnp mutant strains (Hautala, J.A , Bassett, C.L., Giles, N.H. and Kushner,S.R. ~1979] Proc. Natl. Acad~
Sci. USA 76:5774-5778).
These studies also demonstrated a 4- to 7-fold increase in plasmid copy number in these mutant strains.
Thus the increase in enzyme-specific activity could be due to increased mRNA synthesis, increased mRNA lifetime, or a combination of both phenomena.
The rop (repressor of primer) gene has been known for some time to control plasmid copy number. In 1980, it was demonstrated that deletion of a non-essential ~0 region of E. coli colEl derived plasmids increases plas-mid copy number. Deletion of this region increased plas-mid ~NA from 4% of chromosomal DNA to 20%. This deletion ~as trans recessive as coinfection of the host with a ~ild type plasmid reduced the copy number of the mutant plasmid. (Twi~g, A.J. and Sherratt, D. [1980] Nature 283:216-218) Recent prior art reports for E. coli expression systems, wherein proteins foreign to the E. coli host are produced, disclose expression levels of about 25 to 3~ 30% of total cellular protein. Simons et al. reported that human interferon gamma was expressed at levels up to 25% of total cellular protein. These workers utili~ed the PL promoter of phage lambda followed by the transla-tional initiator region derived from either phage MS2replicase or the E. coli tryptophan attenuator region sp:
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-~- R126 (Simons, G~, Remaut, E., Allet, B., Devos, Ro and Fiers, ~. [1984] Gene 28:55-64.) ~mann et al. have ex-pressed the lambda repressor as 30% of total cellular protein using the tac promoter system ~Amman, E., Brosius, JO and Ptashne, M. [1983~ Gene 25:167-178).
As stated above this promoter contains the -10 region of the lacUV 5 promoter and the -35 region of the trp promoter (DeBoer, HoA~ Comstock, L.J., Yansura, D.G.
and Heynec~er, H.L. in Promoters, Structure and Function, 10 Pr~eger, New York [1982] 462-481 (R.L. Rodriguez and M.J. Chamberlin eds.) BRIEF SUMMARY OF THE INVENTION
The subject invention concerns novel hybrid pro-teins which are produced with a novel biological system.
The novel biological system comprises a prokaryotic host transformed with novel hybrid plasmids comprising ~-glucuronidase gene DNA (BG) and the desired protein gene DNA. Specifically exemplified herein is the con-struction of novel hybrid plasmids denoted as plasmid pBG9, plasmid pBG5, plasmid pBG3-2, and plasmid pBG3-2~N.
These plasmids comprise ~-glucuronidase gene DNA and pro-tein A DNA. When used to transform a suitable prokaryotic host, there is realized the production of protein A-like rompounds, i.e., compounds which are indistinguishable from native protein A in the key biological function of binding IgC at the Fc region of the molecule. Advan-tageously, the expression of these hybrid proteins by the transformed host is considerably higher than realized with any known prokaryotic expression system. For example, the fusion (hybrid) proteins exemplified herein are pro-duced at levels of greater than 45% of total E. coli cell protein in host cells containing either the lon or the pnp mutation. Also, advantageously, 100% of the expressed hybrid pro-y .sp:

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-5- ~126 tein is found in the soluble cytosolic fraction upon disruption of the host cell. This result is in contrast to the experience of many skilled in the art who have found that relatively high ex~ression (ca. 7%) of for-eign proteins in E. coli resulted in production of an insoluble and inactive protein.
Plasmid DBG3-2~N exemplifies the ultimate of the ultrahigh prokaryotic expression system. Hosts trans-formed with this plasmid express >60~ of the desired fusion protein. This ultrahigh level of expression is achieved by partially or totally deleting, or otherwise inactivating, the rop gene by constructing a ~Nde dele-tion in plasmid pBG3-2. This procedure can be used on any plasmid derived from the E. coli colEl plasmid 1~ usuable in the subject invention since all of these plasmids contain the ~ region. Examples of such plas-mids are pBR322, pBR325, and pHC79.
Plasmid pBG3-2~N can be used to make a BG/protein A fusion protein containing 18 amino acids of BG-derived sequences and exhibiting protein A activity,i.e., binding IgG at the Fc region of the molecule.
It is surprising that the E. coli host transformed with the novel hybrid plasmids of the subject invention e~presses the fusion BG/protein A product in ultrahigh `25 amounts in view of the known fact that BG is expressed in minute amounts by its native E. coli host. It is believed that this low level expression of BG by native _. coli has led persons skilled in the art away from using BG Dromoter DNA in prokaryotic ex~ression systems.
Rather, the lac and tr~ promoters have been exten-sively used in Drokaryotic ex?ression systems.
The expression system of the subject invention, as exemplified by fusion to the protein A gene or frag~ents thereof, can be used, advantageously, when fused to other ~enes encoding other useful proteins, e.g., interferons, , ~. . ~ :. ::, : .

interleukins, insulins, growth hormones, and indus-trial enzymes, e.g., amylases, proteases, and sugar isomerases, by fcllowing the procedures disclosed herein and attendant procedures known in the art.
DESCRIPTION OF THE DRAWINGS
FIGURE 1: This drawing depicts the construction of an intermediate plasmid from plasmid pAc37.
Plasmid pAc37 comprises the protein A gene and the entire DNA of pBR322.
FIGURE 2: ShOW1 are the restriction maps with gene DNA inserts for plasmids pBG101-41 and pBG9.
The BamHl sites which are not re~enerated during the cloning are marked (BamHl).
FIGURE 3: The construction of plasmid pBG5 from 1~ plasmid pBG9 is shown.
FIGURE 4: The construction of plasmid pBG3-2 from pBG5 and plasmid pBR325 is shown.
FIGURE 5: The construction of plasmid pBG3-2~N from plasmid pBG3-2 is shown.

DETAILED DISCLOSURE OF THE INVENTION
-Before detailing the construction and identity of the novel plasmids, proteins, and expression system of the subject invention, there is disclosed the Materials and Methods employed.
(1) Plasmid DNA preparation - :. ,, . ~ .

~à5~77 Procedure used for large scale preparation of plasmid DNA was essentially as follows: A 250 ml culture was grown to Log ~hase, am~lified with chlor-amphenicol at O.D. 0.6 to 0.7 (or alternatively with no chloramphenicol addition) and grown overnight.
Cells were pelleted at 6K, 20 min, JAl4*rotor, and resus-pended in 6 ml glucose buffer ~50 mM glucose, 25 mM tris, 10 m~l EDTA). Cells were incubated 10 min ac room temp in the presence of l ml of 20 mg/ml lysozyme freshly made;
placed on ice with the addition of 13.8 ml 170 SDS in 0.~ N NaOH for 5 min, and kept on ice an additional 15 min wi~h 7 ml 5 M KAC (pH 5.0-5.5). ~ebris was pelle~ed at 10K ~or l0 min an~ supernate extracted once with an equal vol~ne of phenol-chloroform-isoamyl alcohol (25:24:1, TE saturated, 0.1% 8-hydroxyquino-line). Following precipitation with 0.6 vol. isopropyl alcohol, DNA was purified over CsCl gradients.

~2) Restriction enzyme digestion and isolation of desired fragments Digestions were carried out according to sup-pliers' instructions. Separation of fragments was achieved by agarose gel electrophoresis (described below). Electro?horesed DNA was purified and concen-trated by passing over Elu-tip~columns (Schleicher:
and Schuell, Keene, NH) according to supplier's instructions, followed by precipi~tation in 2.5 volumes EtOH with added carrier tRNA.

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(3) Minilysate plasmid analysis Transformed cells were inoculated into 1 ml of L-broth supplemented with either 10 ~g/ml tetracycline or 50 ~g/ml ampicillin and grown for 3-5 hr at 37C. The cells were collected by c~entrifugation at lO,OOOxg for 15 min then resuspended in 50 ~l of STET~ buffer (8% suc-rose, 5% Triton* X-100, 50 mM EDTA, 50 mM Tris*-HCl pH 8.0).
50 ~l of lysozyme solution (2 mg/ml in ST~T~ buffer) was added and ~he tubes were incubated for 4 min at room temperature, then heated to 100C for 3 min. The tubes were then cooled to 0C on ice. After 5 min at 0C, the insoluble material was removed by centrifugation at lO,OOOxg for 15 min. An equal volume of ice cold isopropyl alcohol was added to the supernatant and the tubes were placed at 70C for 5 min. The DNA precipitate was collected by centrifugation at lO,OOOxg for 10 min and resuspended in 10-25 ~l of TE buffer (10 mM tris-Cl, 0.1 mM EDTA pH 8.0).
Restriction digest of the DNA was performed as described above using 5 ~l of plasmid solution in a final volume of 15 ~l containing 6.7 ~g/ml of RNase A.

(4) DNA ligations T4 ligase was used for both sticky and blunt end ligations, and was in each case present in excess (200 units/~g DNA). For sticky ends, incubation time was 2-4 hr at 16C and for blunt ends the time was increased to 16 hr. For standard vector/insert ligations, insert was present in a 5-fold molar excess with 0.02 pmoles o~ vector and 0.1 pmoles of insert in a ~0 ~l reaction volume. F~r the generation of deletion mutants by a unimolecular recircularisation reaction ? plasmid ~ trade mark ;~
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(5) Transformation Fresh overnight cultures were diluted in L-broth and allowed to grow at 37C with agitation until an A600 of 0.3 was obtained. The cells were chilled on ice, ~hen collected by centrifugation (10 min at 4100xg).
The cells were resuspended in l/2 the original volume or ice cold 50 mM CaC12 and incubated on ice for 20 min.
The cells were again collected by centrifugation as above and resuspended in ice cold 50 mM CaC12 (1/2S the original volume). 0.1 ml of the cell suspension was mixed with 1-lO ~l (50-100 ng) of DNA plasmid solution and allowed to sit for 30 min at 0C. The cells were then lleated to 37C for 2 min and plated on L broth plates containing 1.5% agar and either 10 ~g!ml tetra-cycline or 50 ~g/ml chloramphenicol when pBR325 deriva~-tives are transformed. The plates were incubated overni~ht at 37C. Transformation efficiencies of lx106 colonies per ~g plasmid DNA were routinely ob-~5 served.

t6) Agarose electrophoresis D~A fragments were isolated by gel electrophoresis in 0.87~ agarose in 2X tris-borate buffer (178 mM tris, 17?3 mM boric acidj 5 mM Na2EDTA pH 8.4). Analytical and preparative gels were run in a horizontal gel box at : . : :,:. ~ . :
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'77 60 volts submerged in electrophoresis buffer (lX tris-borate). DNA bands were visualized under UV light by including 5.0 ~g/ml e~hidium bromide (EtBr) in the gel.
A slice containing the desired DNA band was cut from the gel and che DNA recovered by electrophoresis in lX
tris-borace buffer in a dialysis tube (1/2 in.diameter) concaining 0.5-l~0 ml of buffer. Electrophoresis was carried OUt for 30 min ac lO volts or until the s~ained macerial was loca~ed against the side of the dialysis tubing~ The gel slice was removed from the dialysis ba8 and the DNA recovered by repeatedly flushing the bag with tris-borate buffer. NaCl was added to the DNA
solution to a final concentration of 1 M and the ethi-dium bromide and agarose gel impurities were removed by two e~tractions with phenol saturated with tris bora~e buffer. The ~henol was removed by two extrac-tions with ether and the ~urified DNA was recovered by precipitation with 1/50 volume 5 M NaCl and 2.5 volumes cold ethanol. The precipitation reaction was carried ou~ at -70aC for 15-20 min. The preci~itated DNA was recovered by centrifugation at lO,OOOxg for 15 min. Yield of recovered fragment was assayed by direc~ comparison o~ eLhidium bromide fluorescence with pure DNA stan-dards. Typically, 50% recoveries were ob~ained~with the ~5 yield decreasing as fragment size increased.

(7) Protein A radioassay Protein A activity was determined by coating Dynatech Immunolon~(Dynatech Diagnostics, Inc., South Windham, ME) 1 microtiter wells with 50 ~1 o a 1:1OJOOO dilution of normal rabbit serum (NRS) and rdc~n13 r ~

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incubating at room temperature for 4 hr. The NRS was shaken from the wells, which were then blocked with 1%
ovalbumin in phosphate buffered saline (OVA/PBS) by incubation for 1 hr at 4C. The wells were emptied;
then 25 ~l samples containing between 0.1 and 1,000 ng protein A were added to each well. A standard curve utilizing commercial protein A was run in each assay.
All dilutions were in OVA/PBS. 25 ~1 of 125I-protein A
(6,000 ~pmJ in OVA/PBS was added to each well and the plates were incubated for 16 hr at 37C in a sealed plastic container containing a small beaker of water.
Following incubation, the wells were aspirated and washed 3X with PBS and once with water. The wells were dried and counted for 2 min in 2 ml Aquasol (New England Nuclear Corp., Boston, MA) in a Beckman~ model LS7000 beta counter (Beckman Instruments, Inc., Fullerton, CA).
(8) Protein A rocket immunoelectrophoresis Protein A concentration and activity was determined by rocket immunoelectrophoresis in a 1%
agarose gel containing 31 ~g/ml human IgG in tris-glycine pH 8.6 buffer (3.75 g/l tris base, 7.5 g/l glycine).
Protein A standards between 0.25 and 1.0 ~g were run on every gel. Electrophoresis was allowed to proceed for 3 hr at 400 volts using tris-glycine as an electrophor-esis buffer. Following electrophoresis, the gels were dried, then briefly stained with Coomassie blue and destained with 5% methanol, 10% acetic acid.

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(9) Cell homo~enizat on Transformed cells were collected by centrifugation at l~,OOOxg for 5 min at 4C and resuspended in 0.5 vol-umes of HEPES (4~(2-hydroxyethyl)-1-piperazine-ethane-sulEonic acid)/KCl/DTT (dithiothreitol) buffer (6 gm HEPES pH 8.0, 7.5 gm KCl, 0~15 gm DTT per liter). The cell suspension was digested with lysozyme at a final concencration of 300 ~g/ml for 30 min at 37C. The suspension was sonicated by two 5 min pulses at 300 la wacts on ice. Soluble protein was isolated by centri-~ugation at 25,000xg for 30 min at 4C. The supernatant was removed and the preci?itate was suspended in an equal volume of HEPES/KCL/DTT buer. For experiments where total cell protein was run on SDS gels, the cells were solubilized by heating to 100C for 5 min in 5 volumes of S3S-homogenization bufer (50% v/v glycerol, 5~ v/v 2-mercaptoethanol, 5% w/v sodium dodecyl sulfatc, and 0.005 mg/ml pyronine Y).

~0 (~lO)Polyacrylamide ~el electrophoresis and Western anaLysis AlL SDS gels were run by the method of Laemmli (Laemmli, U.K. [1970] Nature [London] 227:680-685). These gels contained a total acrylamide concentration of 12%. Slab gels were l.5 mm wide, run in an electro-phorecic apparatus obcained from Hoefer Scientific Inscrumencs (San Francisco, CA). Tube gels were run in

6 mm i.d. x lO cm glass tubes without a stacking gel.
Western blots were performed on nitrocellulose i1ters.
Protein was transerred to the filters at 200 mA for 12 hr. The filters were blocked for 4 hr with 0.1%
bovine serum albumin (BSA) in phosphate buffered saline (PBS) at room temperacure and h~bridized with ei~her ~ ` ~
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lO uCi of [Il25]-IgG (NEN) or lO0 ul of rabbi~ IgG
conjugated with peroxidase a~ room temperature over-night with agitation. The blots were then washed 4X
with PBS and exposed to Koda~*XAR-5 x-ray film or developed with 25 mg diaminobenzidine in lO0 ml PBS with 25 ~1 H22 ' (ll) Measurement of protein A content in cloned cells Following fermentation, cells were homogenized in ~0 mi~l tris-HClpH 8.3 containing 0.5~ Triton X-lO0 by vortexing with glass beads or in a DyanoMill~model KD~-pilot bead mill (obtained from Impandex, Maywood, N.J.) operated at maximum speed and charged with 0.2 mm dia-meter glass ~eads. The homogenate was clarified by centrifugation at 16,000xg for 30 min and the supernatant protein concentration measured by the Lowery ~rotein assay or by biuret. Pro~ein A concentration was mea-sured by rocket immunoelectrophoresis against human IgG.
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(12) HPLC purification of proteins Protein A and protease K were purified or assayed by HPLC using a Beckman model 360 gradient machine (Beckman Instruments, Inc.) fitted with a Waters ~5 ~Bondapak C18 column (Waters Associates, Milford, MA).
Protein A was purified by a linear gradient between I0 m~l sodium phosphate pH 7.2 (buffer A) and 60% v/v iso-propanol 10 mM phosphate (buffer B). The column was eluted at a flow rate of l ml/min with a linear gradient between 0 and 100% buffer B over 80 min. Protease K
was purified and protein A assayed in a similar manner except that buffer A contained 0.1% trifluoroacetic acid (TFA) and buffer B was 0.08% TFA in acetonitrile.
The column was eluted at a flow rate of 2 ml/min by a linear gradient between O and 60% buffer B over~60 min.
I r~c ~k ,, ~2~5~ 7 (13) Fermentation Fermentation was performed in a 201 Chema3ec~
fermentor (Chema~ec, Inc., Woodbury, NY) fitted with do2 and pH control. Recombinant cells were grown at a do2 of 50~ (air=100%) at the T?H indi-cated. pH was adjusted by addition of 5 M NH40H or 5 ~I H~SO4 as required Foam was controlled b~ addition of antifoam B~(E.I. du Pont De Nemours ~
Co., Inc., Wilmington, DE). Fermentation temperature was 37C; all fe~mentations were conducted with a final volume of 9.51.
(14) Bacterial s~rains and media The source and genotype of all bacterial strains used are listed infra. All strains were main~ained and gro~ using YT medium (8 gm/l tryptone, 5 gm/l yeast extract, and 5 gm/l sodium chloride).
(15) Chemicals Nitrocellulose was obtained from Schleicher and Schuell (Keene, NH). Growth media were obtained from 2~ Difco (Detroit, MI). Acrylamide was obtained from Accurate Chemical ~ Scientific Corp., ~T~estbury, NY).
Protein A standard was obtained from Pharmacia (Pisca-taway, NJ). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
(16) Cultures (A) Bacterial--All E. coli strains disclosed herein are _. coli K-12 derivatives.
Strains Relevant Genotype Repository Number .
~. coli F ,Gal ,Thi ,endA NRRL B-15129 MS371 sbcB,hsd~4 Deposited Aug. 18, 19'32 and now avail-able to the public . upon request to the NRRL repository.
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(A) Bacterial cultures (cont.) Strains Relevant Genotype Repository Number .
SG20251 F ,ara D139,1ac, NRRL B-15918 lon-lOO,InlO::c~s E, Deposited on str A,thi Dec. 12, 1984.
-PR13 F ,pnp-13,rna-19, Can be obtained from thr-l,leu B6,thi-1, deposited cultures 1(~ lac Yl,~y~-7,mtl-2, listed below by mal Al,str A132, (=rps standard ~rocedures.
L132) (B) Bacterial host containin~ ~la_mid Repository Host Number E. coli MS371(~Ac37) NRRL B-15127 De~osited on Aug. 18, - 1982 and now available to the ?ublic upon request to the NRRL culture repository.

MS371(pBG101-41) - NRRL-B-15905 De30sited on Nov. 1, 1984 ~5 PR13(pBG9) NRRL B-15907 Deposited on Nov. 20, 1984 PR13(pBG5) NRRL B-15908 Deposited on Nov. 20, 1984 PR13(?BG3-2) NRRL B~15909 3~ Deposited on Nov. 20, 1984 PR13(pBG3-2~N NRRL B-15910 Deposited on Nov. ~0, 1984 (C) Plasmids - Plasmid pBR322 is a well-known and available ~lasmid. It is maintained in the E. coli host ATCC
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.: . ." , ~ : , ~ 77 37017. Puriried pBR322 DNA can be obtained as de-scribed in Bolivar, F., Rodriquez, R.L., Greene, P.J.
Betlach, M.C., ~eyneker, H.L., Boyer, H.W., Crosa, J.H.
and Falkow, S. (1977) Gene 2:95-113; and Sutcliffe, J.G. (1978) Nucleic Acids Res. 5:2721-2728. Plasmid , , .............. _ ..... . .. ..
pBR325 is also a well-known plasmid. It can be obtained from BRL Inc., P.O. Box 6009, Gaithersburg, MD 20877.
NRRL B-15907, NRRL B-15908, NRRL B-15909, NRRL
B-15910, and NR~L B-15918 are available to the public upon the grant of a patent which discloses these acces-1~ sion numbers. It should be understood that the availability of these deposits does not constitu-te a license to practice the subject invention in derogation o~ patent rights granted for the subject invention by governmen-tal action. The culture de~osits are in the permanent 1~ collection of the Northern Regional Research Laboratory (NRRL), U.S. Department of Agriculture, Peoria, Illi-nois, USA.
There are other well-known E. coli hosts which can be used instead of E. coli PR13, for example, E. coli o ~IS371, HB101, and E. coli GMS407 (Novel, M. and Novel, G.
~1973] Mol. Gen. Genet. 120:319).
Further, other prokaryotic hosts which can be used are microbes from the genera Salmonella, Pseudomonas, Bacillus, Streptomyces, and the like.
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(17) Isolation of recombinant plasmid DNA from trans-formed host Recombinant plasmid DNA can be isola~ed from its prokaryotic host by well-known procedures, e.g., using cleared lysate-isopycnic density gradient procedures, and the like.

(18) DNA sequencin~
DNA sequence determination was carried out as described by Maxam and Gilbert (Maxam, A. and Gilbert, .
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W. [1977] Proc. Nat'l. Acad. Sci. USA 74:560) and Heidecker et al. (Heidecker, G., Messing, J., and Gronenborn, B. [1980] Gene 10:69) Construction of hybrid protein genes The construction of the hybrid protein genes, e~emplified herein as representative of the invention, was initiated with the use of plasmid pBG101-41. This plasmid contains approximately 6 kb of E. coli 3-glu-curonidase gene DNA inserted at the BarnHl site of plasmidpBR322. Plasmid pBG101-41 was cut with restriction endonuclease BamHl and blunted by brief treatment with Bal-31 exonuclease. This exonuclease treatment removed 1~ bases and left a blunt end.
DNA for insertion into the cut and blunted pBG101-41 was obtained from plasmid pAc37 which contains the Staphylococcus aureus protein A gene in pBR322. See -Figure 1 of the Drawing.
The cut and blunted olasmid p~G101-41 was ligated with the blunt-Cla~ protein A fragment to give hybrid plasmid pBG9. Plasmid pBG9 contains 501 nucleotides coding for the N-terminal 167 amino acids of the ~-glu-curonidase protein fused to the protein ~ gene. See Figure 2 of the Drawing.
2~ Hybrid plasmid pBG5 was constructed from hybrid plasmid pBG101-41 and hybrid plasmid pBG9. See Figure 3 of the Drawing. Plasmid pBG101-41 was cut with BamHl and ~hen digested with Bal-31 exonuclease (IBI-ast Bal-31). The resulting DNA was digested with Clal:
and insert DNA, prepared as discLosed infra, was liga~ed.
The insert DNA for the above ligation, containing the mature protein A coding sequences, was prepared from hybrid plasmid pBG9 by cutting this plasmid with the restriction enzyrnes Clal and Fnu4Hl.

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The insert and vector DNA were ligated and trans-formed into _. coli strain PR13, and plasmid DNA was prepared from the transformants. A clone, labelled pBG5, contained the predicted restriction profile.
Sequence analys s of this clone by the standard M13 method revealed ~hat 18 amino acids of the BG coding sequence remained.
Hybrid plasmid pBG3-2 was constructed from plasmid pB&5 and plasmid pBR325. See Figure 4 of the Drawing.
Plasn)id pBG3-2 contains the same DNA as plasmid pBG5 except that pBG5 contains pBR322 DNA and pBG3-2 contains p~R325 DNA; also, pBG3-2 contains a stop codon linker at the ClaI site at the end of the proteinA 8ene D~A.
The constructed linker segment of DNA contained stop codons in all three reading frames. It was inserted into the CIaI site in the pBG3-2 construction to insure that the final hybrid protein product did not contain any pBR325-derived amino acids.
Increased expression of the hvbrid protein encoded ~0 by the fused gene in plasmid pBG3-2 was obtained by constructing a ~Mde deletion, i.e., by removing the DNA
bet~een the Nde site in pBR325 and the Nde site on the sequence. This deletion removed the bulk of the ~
~ene in pBR325, as well as the first 230 bases of the BG
~5 promoter re~ion. This construction is identified as plas-mid DBG3-2~N. When an E. coli host is transformed with pBG3-2/~N, the host ex~resses`?rotein A at levels >6~% of total E. coli protein. In comparison, protein A is ex-pressed in E. coli at iO% of total cellular protein in host cells containing the Plasmid pBG3-2.
Utility of protein A
Protein A is widely used as an immunoabsorbent in a variety of diagnostic and basic research test systems.
See U.S. Patent No. 4,322,274. Recent interest in 3~ applications of protein A has centered around its ... .
.
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-19- Rl26 possible clinical use in anticancer treatment. Sensi-tized peripheral blood lymphocytes, normally responsible for cytoto~icity of tumor cells, are hypothesized to be inhibited in this function by serum blocking factors which are presumed to consist of specific antigens, antibodies, antiglobulins, and immune complexes. See ~arnes, B.C. (1981) Cancer Bull. 33:278. These "block-ing`' factors can be removed from sera of tumor-bearers by absorption to S. aureus, Cowan I cells which contain lG protein A, and thus allow cell-mediated tumor cell to~icity to proceed in in vitro test systems. See Steele, G , Ankers~, J., and Sjogren, H. (1974) Int. J. Cancer 14:83. Protein A also activates polyclonal antibody synthesis independent of its IgG binding activity. See Sjodahl, J. and ~oller, G. (1979) Scand. J. Immunol.
10:593.
Extensive tescing of protein A as an anticancer agent has been inhibited by the high cost of the material and by the presence of impurities in some protein A
~0 preparations. Should the cost of protein A preparations be significantly reduced and the purity improved, then further clinical testing of protein A for anticancer uses would proceed more rapidly.
Having the data disclosed herein, those skilled _5 in the art can readily appreciate the identity of other equivalent nucleotide sequences coding for molecules with substantially the same protein A-like biological activity. Thus, the scope of the subject invention includes not only the specific nucleotide sequence depicted above, but also all equivalent nucleotide sequences coding for molecules with substantially the same identifiable protein A-like biological activity.
The term "equivalent" is being used in its ordinay patent usage here as denoting a nucleotide seque~ce ' . :

~2~ 7 which performs substantially as the nucleotide sequence iden~ified herein to produce molecules with substan-tially the same identifiable protein A-like biological ac~ivity in essentially the same kind of hosts. Within this definition are subfragments of the protein A-like material which have the ~roperty of binding to IgG
at the Fc region, or sub~ragments which have polyclonal B-cell activatirg activity. Plasmid oAc37, disclosed in Example 1, contains the entire nucleotide sequence l~ coding for the ainino acid sequence of Staphylococcus aureus protein A. This sequence, which is shown in Chart A, enables persons in the art to obtain cloned nucleotide sequences coding for identifiable protein A-like material and identifiable subfragments of protein l~ A-like material, as defined above. The identifiable orotein A-like material of the subject invention, and identifiable protein A-like subfragments thereof, can be used in the same manner as protein A, disclosed above.

~0 Following are examoles which illustrate procedures, including the b~st mode, for practicing the invention.
These examples should not be construed as limiting.
~ll percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
2~
Example l--Cons~ruction of Hybrid Plasmid pBG9 from Plasmid pBG101-41 and Plasmid pAc37 and Exoression of Fusion Protein A Product The pLasmid pBG9 containing the ~-glucuronidase promoter and the 3-glucuronidase-protein A hybrid gene was constructed from the plasmid pBG101-41 and the blunt-ClaI protein A fragment described herein. Plas-mid pBG101-41 was opened at the unique BamHI site (lo-cated 179 amino acids after the initiation methionine) and blun~ed by brief treatment with Bal-31 exonuclease .

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(as described by manufacturer). This exonu-clease treatment removed 36 bases (12 amino acids) and left a blunt end. The plasmid was further cut with ClaI at the unique site in plasmid ~BR322.
Plasmid pAc37 contains the protein A gene in pBR322.
Plasmid p~c37 was digested with Rsa which cleaves the pl-otein A gene at ~osition 65 and 1264 after the TTG
s~art codon (T=li. The 1199 base r,air Rsa fragment was isolated by agarose electrophoresis. ClaI linkers (New England Biolabs, Beverly, MA, sequence CATCGATG) ~ere fused to the isolated Rsa fragment. This construc-tion was cut with ClaI and inserted into the ClaI site of pBR322 to form an intermediate plasmid designated pAl. Plasmid pAl was partially digested with ClaI and the ClaI sticky end filled in in a reaction containing 2 mM each of the 4 deoxynucleotide triphosphates and 5 units of the Klenow fragment of E. coli DNA polymerase l in 25 ~l of 50 mM tris-Cl pH 7.2, 10 mM Mg2SO4, 0.1 m~l DTT, 50 l~g/ml BSA and l ~g of the restriction frag-o ment. The fill-in reaction was incubated for 20 min at 22C and s~opped by heat inactivation at 70C for lO min. The plasmid was then digested with Sa~ and the 1826 base pair fragment isolated by agarose electrophor-esis. This fragment was further cut with Clal and in-serted into the cut plasmid described above. (See Figure l of the Drawing.) The D~A sequence of Glasmid pBG9 and the amino acid sequence of the fusion protein ex?ressed by E.
coli PRl3(pBG9) is shown in Chart B.
Plasmid pBG9 and Plasmid pBG101-41 and Expression of Fusion Protein P Product The plasmi~ pBG101-41 consists of pBR322 which has been opened at the BamHI site with insertion of the SauI partial sequences containing the BG promoter and BG coding domains. Plasmid pBG101-41 was cut with BamHI, . `. , , -,. :

3~77 which cleaves this plasmid at a site 179 amino acids after the methionine start codon, then digested with Bal-31 exonuclease (IBI-fast Bal-31) at an enzyme concen-tration of 20 U/ml and a ~NA concentration of 100 ~g/ml.
The reaction was allowed co proceed at 30C. At 10 min, 15 min, and 20 ~.in one-third of the digest was removed and the reactior. halted by addition of EDTA to 20 mM, followed by free~ing at -80C. The time points were individually extracted with phenol-ether and precipi-tated with ethanol. The DNA was digested with ClaI,whichcuts in the unique site in pBR322; then insert DNA was ligated.
Insert DNA containing the mature protein A coding sequences was prepared from the plasmid pBG9. This plasmid was cut with the restriction enzymes Clal and Fnu4Hl Restriction endonuclease Fnu4Hl cuts the protein A gene one base to the 5' end of the signal peptide cleavage point and ClaI cuts the gene in the C-terminal repeati~g domains. This ClaI site was con-structed by liga:ing a ClaI linker at the Rsa sitelocated 2S4 base pairs from the 3' end of the protein A gene.
Insert and vector DNA were ligated together in a 4:1 insert to vector ratio in a reaction containing 20 ~5 ~g/ml vector DNA. The T4 ligase-catalyzed reaction was allowe~ to proceed overnight at 15C; then ligase was inactivated by heating to 70C for 15 min. The reaction mixture was digested with Xho (which cuts at a unique site in the BG protein) to prevent transformation of any plasmids containing a BG deletion. The reaction mixture was transformed into E. coli strain PR13 and plasmid DNA was prepared from the transformants. A clone, labelled pBG5, contained the predicted restriction profile. Sequence analysis of this clone by the M13 method revealed that 18 amlno acld~ of the BG coding :..,:, .. :, ,, :':.,,,.~. ', ' ~ ' ; : ' , " " :

5~)~7 sequence remained. (See Figure 3 of the Drawing.) The DNA sequence of plasmid pBG5 and the amino acid sequence of the fusion protein expressed by E. coli pRl3(pBG5) is shown in Chart C.

Exam~le 3--Construction of Hybrid Plasmid pBG3-2 from Plasmid pBG5 and Plasmid pBR325 and Expression of Fusion Protein A Product Plasmid pBR325 was digested with ClaI and SalI and 1~ the 5368 base pair fragment containing the bulk of the plasmid coding sequences was isolated by agarose electro-phoresis. Plasmid pBG5 was also diges.ed with ClaI and SalI and the 2000 base pair fragment containing the BG
promoter and the protein A coding sequences was isolated by agarose electrophoresis. These two DNA fragments were mixed ina~ equal molar ratio at 30 ~g/ml per ~ragment and ligated with T4 ligase. The resulting product was digested with ClaI and the resulting linear molecule of _ 7.4 kb was isolared by agarose electrophoresis. A
~0 linker DNA fragment containing the stop codons, ?repared as described in Example 4, was added in large molar excess and the reaction ligated with T4 ligase overnight at 15C. The closed circular plasmid was digested with ClaI and SmaI to linearize plasmids ~5 containing multiple or no stop linkers, then trans-formed into _. coli PR13. ~See Figure 4 of the Drawing.) The DNA sequence of plasmid p~G3-2 and the amino acid sequence of the fusion protein expressed by E. coli PR13(pBG3-2) is shown in Chart D.
Example 4--Construction of A Sto~ Linker A linker seg~ent of DNA containing stop ~odons in all three reading frames was inserted into the ClaI
site in the pBG3-2 construction to insure that the final product did not contain any pBR-derived amino - .~ ,~ . , , 5~77 -2~- R126 acids. A synthetic DNA segment with the sequence CGGGCGCGCTAGCTAGCTAGCGCGCC was synthesized using an Applied Biosystems DNA synthesis machine Model 380A
(Foster City, CA) by the procedure suggested b~ the manufacturer. This sequence is self annealing and yields the double stranded DNA fragment:
C G G G C G C G C T A G C T A G C T A G C G C G C C
C C G C G C G A T C G A T C G A T C G C G C G G G C
which contains the stop sequences CTAGCTAGCTAG and the BssHI site: GCGCGC at both ends of the triphasic stop Example 5--Construction of Plasmid pBG3-2~N from Plasmid pBG3-2 Plasmid pBG3-2 was digested with restriction endonuclease Nde and the cut plasmid extracted with phenolether and precipitated with ethanol. The plasmid was religated at dilute DNA concentration (12 ~g/ml) to favor intermolecular recircularization without incorpor-ation of the Nde fragment to give plasmid pBG3-2~N.
The reaction mix was transformed into E~ coli PR13 and the colonies assayed by minilysate analysis. See Figure 5 of the Drawings.
Example 6--Transformation of plasmids pBG3-2, pBG3-2~N, pBG9 and pBG5 into E. coli PR13 or E. coli SG23251 E. coli PR13 or E. coli SG20251 were harvested from fresh overnight cultures grown as described in (5) Transformation.
The cells were made competent for transforma-tion by treatment with CaC12 as described.
Plasmid DNA was prepared from cells harboring the plasmid by the methods described in (1) Plasmid DNA preparation.
0.1 ml of the competent cells were mixed with 50-100 ng of plasmid DNA for 30 min at 0C. The cells sp: ' :: : . : ....... ;
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~.2~t~7 were heated to 37C for 2 min then pla-ted on L-broth ~lates containin~ 1.5% agar and either 10 ~g/ml tetra-cycline or 50 ~g/ml chloramphenicol when pBR325 deriva-tives are transformed. The plates were incubated overnight at 37C. Transformation efficiencies of 1~106 colonies ~er ~g plasmid DNA were routinely ob-served.

E~am~le 7--Fermentation of E. coli PR13(pBG3-2?
1~
E. coIi PR13(pBG3-2) can be grown by any of a num-ber of methods familiar to those skilled in the art.
This organism will grow on any complex medium capable of supporting the growth of E. coli and on any defined medium if such defined medium cont~ins sufficient growth factors and metabolites necessary to support cell growth. In general these defined media comprise those capable of supporting the growth of E. coli if chey contain the amino acids threonine and leucine.
Production of recombinant protein by this organism is subjec~ to catabolite repression. Thus, when ~rotein production is desired, care must be taken that the growth medium does not contain glucose or any substance capable of causing catabolite repression. Catabolite n~ repression in E. coli is mediated by an intercellular -- .
decrease in the levels of cAMP. Thus, this organism can be grown in che presence of growth media containing glucose if those media contain a high level of cAMP, typically 4 mM, or if those media contain high levels of a lipid soluble cAMP derivative, for example, dibuteryl-cyclic AMP at a concentration of about 10 ~M.
In general, high levels of protein A can be pro-duced by preparing an inoculum from a frozen stock of E. coli PR13(pBG3-2), which was streaked on YT/Cm ,::, , ,: ,: . :,. . ~ ,.. . .
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...:, ~ '77 medium and grown overnight. Y~ contains 8 g/l yeast e~tract, 5 g/l tryptone and 5 g/l NaCl. YT/Cm contains 50 mg/l chloroamphenicol. A colony was picked from this plate and inoculated into 10 ml of YT/Cm which was grown at 37C for 6-12 hr then inoculated directly into the fermenter.
E. coli PR13(pBG3-2) was grown in a 201 Chemapec fermenter (Chemapec, Woodbury, NY) charged with 9.8 1 of 5 gm/l yeast extract and 5 gm/l tryptone. The dis-solved oxygen concentration is maintained at about 50%~air--100%) and the pH was maintained at about pH 6.8 by automatic addition of 5 M NaOH or 5 M H2S04. The normal inoculum volume is ab~ut 10 ml. With this inocu-lum, the fermenter can be harvested after 9 hr of growth.
When cells are grown in this manner, 46% of the total E. coli derived protein produced in the fermenter is protein A.
Evidence demonstrates that cloned protein A is e~pressed in an active form. A Western blot probed with [125I] labelled rabbit IgG shows that the hybrid protein has IgG binding activity even after treatment with hot SDS solution and electrophoresis in SDS poly-acrylamide gels.
The specific activity of soluble protein A ex-tracted from the pnp- host strain was determined by radioassay (see (6) Protein A radioassay). This assay demonstrated that cell cytosol had protein A activity which was 35% of the specific activity of pure commercial material. Protein A concentration in this cytosolic pre-paration was determined to be 35% by SDS gel electro-phoresis, indicating that the cloned material has essen-tially identical specific activity with the naturally occuring protein.

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Example 8--Fermentation o~ E. coli PR13(pBG3-2~N) .
When the recombinant organis~ is grown in a fermenter as described in (13) Fermentation, like plas-mid pBG3-2, plasmid pBG3-2~N is subject to catabolite repression. The media and conditions described for E. coli PR13(pBG3-2) can be used to grow this organism as well. Surprisingly, E. coli containing plasmid pBG3-2~N produces an extraordinarily high level of re combinant product.
The following table shows the protein A expres-sion levels of pBG9, pBG3-2 and pBG3-2~N:
Protein A Expression Levels No. of BG Expression Amino Acids Level*
pBG9 168 46%
pBG3-2 18 50%
pBG3-2~N 18 73%

* Protein A as percent of soluble cell protein. Protein A content is determined by Rocket immunoelectrophoresis and total protein by biuretO

Example 9~ olation of Host Transformed with a Plasmid The host microbe, e.g., E. coli PR13~ can be re-covered minus the plasmid, e.g., pBG99 with which it was transformed, by standard procedures. For example, the transformed host can be grown in YT medium containing 0.01% w/v SDS to eject the plasmid from the host. Host cells without plasmid can be screened because of the loss of resistance to chloramphenicol and/or ampicillin.

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As is well known in the art, the amino acid sequence of a protein, e.g., protein A, is determined by the nucleotide sequence of the DNA. Because of the redundancy of the genetic code, i.e., more than one cod-ing nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleo-tide sequences can code for a particular amino acid.
Thus, the genetic code can be depicted as follows:
Phenylalanine (Phe) TTK Histidine (His) CAK
10 Leucine (Leu) XTY Glutamine (Gln) CAJ
Isoleucine (Ile) ATH Asparagine (Asn) AAK
~ethionine (Met) ATG Lysine (Lys) AAJ
Valine (Val) GTL Aspartic acid (Asp) GAK
Serine (Ser) QRS Glutamic acid (Glu) GAJ
Proline (Pro) CCL Cysteine (Cys) TGK
Threonine (Thr) ACL Tryptophan (Try) TGG
Alanine (Ala) GCL Arginine (Arg) WGZ
Tyrosine (Tyr) TAK Glycine (Gly) GG~
Termination signal TAJ
~0 Termination signal TGA
Key: Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of mRNA, having a 5'-end on the left and a 31-end on the right. All DNA sequences given herein are those of the strand whose sequence corresponds to the mRNA
sequence, with thymine substituted for uracil. The letters stand for the purine or pyrimidine bases forming the deo~ynucleotide sequence.
A = adenine G = guanine ~ = cytosine T = thymine X = T or C if Y is A or G
X = C if Y is C or T
Y = A, G, C or T if X is C
Y = A or G if X is T

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W = C or A if Z is A or G
W = C if Z is C or T
Z = A, G, C or T if W is C
Z = A or G if W is A
QR = TC if S is A, G, C or T
J = A or G
K = T or C
L = A, T, C or G
M = A, C or T

~0 The above shows that the novel amino acid sequence of the fused protein A product ? and other useful proteins, can be prepared by equivalent nucleotide sequences encoding the same amino acid sequence of the proteins. Accordingly, the subject invention includes such equivalent nucleotide sequences. In addition it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T. and Ké2dy, F.J. [1984] Science 223:249-255).
~d The work described herein was all done in conform-ity with physical and biological containment requirements specified in the NIH Guidelines.

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)7 7 -3a-CHART A: Nucleotide sequence coding for the amino acid sequence of Sta~hylococcus aureus Protein A.
CHART B: Shown is the DNA sequence of hybrid plasmid pBG9 and the amino acid sequence of the expressed fusion protein.
CXART C: The DNA sequence of hybrid plasmid pBG5 and the i~mino acid sequence of the fusion protein e~pressed is shown.
C~RT D: Showr is the DNA sequence of hybrid plasmid 1~/ pBG3-2 and the amino acid sequence of the expressed fusion protein.

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CY~RT B--pBG9 (pa~c 1) EcoRV
Sau3A Rsa I Taq I
GAT CTG ACC TAC GGT GTA CTG &CC GAT ATC GAA GCG GAA GAC

Dde I
CTG GCG CGT GAA GCG TCG TTT GCT CAG GGA TTA CGC GCG ATG

ATT GGC GGT ATC TTA ACC GCA TCC TGA TTC TCT CTC TTT TTC

GGC GGG CTG GTG ATA ACT GTG CCC GCG TTT CAT ATC GTA ATT

Eco RI
TCT CTG TGC AAA AAT TAT CCT TCC CGG CTT CGG AGA ATT CCC

Nde I
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Taq I
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.

Hinc II Aha III Sau3A
GCT TCT GTC AAC GCT GTT TTA AAG ATT AAT GCG AT.C TAT ATC

Sau3A
ACG CTG TGG GTA TTG CAG TTT TTG GTT TTT TGA TGG CGG TGT
: ::
-10 ~ : Nco I
CAG TTC TTT TTA TTT CCA TTT CTC TTC CAT GGG TTT CTC ACA

' 3 (LJ

CHART B (~
Xinc II

~ ---------------- Hpa I
GAT AAC TGT GTG CAA CAC AGA ATT GGT TAA CTA ATC AGA TTA

Hinc II RBS
AAG GTT GAC CAG TAT TAT TAT CTT AAT GAG GAG TCC CTT ATG
Met Taq I
TTA CGT CCT GTA GAA ACC CCA ACC CGT GAA ATC AAA AAA CTC
Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Nru I
Sau3A

Asp Gly Leu Trp Ala Phe Ser Leu Asp Arg Glu Asn Cys Gly Bcl I
Sau3A
ATT GAT CAG CGT TGG TGG GAA AGC GCG TTA CAA GAA AGC CGG
Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln Glu Ser Arg GCA ATT GCT GTG CCA GGC AGT TTT AAC GAT CAG TTC GCC GAT
Ala lle Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala Asp Ala Aso Ile Arg Asn Tyr AIa Gly Asn Val Trp Tyr Gln Arg Fnu4H
. GAA GTC TTT ATA CCG A M GGT:TGG GCA GGC CAG CGT ATC GTG CTG
Glu Val Phe Ile Pro Lys Gly Trp Ala Gly Gln Arg:Ile Val Leu `: :

- : ,, .. .. : . ;.

Rl.~6 CHART B (Page 33 Taq I
CGT TTC GAT GCG GTC ACT CAT TAC GGC AAA GTG TGG GTC AAT
Arg Phe Asp Ala Val Thr His Tyr Gly Lys Val Trp Val Asn Fnu4H

Asn Gln Glu Val Met Glu His Gln Gly Gly Tyr Thr Pro Phe Rsa I
GAA GCC GAT GTC ACG CCG TAT GTT ATT GCC GGG AAA AGT GTA
Glu Ala Asp Val Thr Pro Tyr Val Ile Ala Gly Lys Ser Val CGT ATC ACC GTT TGT GTG AAC AAC GAA CTG AAC TGG CAG ACT
Arg Ile Thr Val Cys Val Asn Asn Glu Leu Asn Trp Gln Thr ATC CCG CCG GGA ATG GTG ATT ACC GAC GAA AAC GGC AAG AAA
' 71 Ile Pro Pro Gly Met Val Ile Thr Asp Glu Asn Gly Lys Lys Fusion site Taq I
AAG CAG TCT TAC TTC CAT GAT TTC TTT AAC TCG ATG ACA TTA
Lys Gln Ser Tyr Phe His Asp Phe Phe Asn Ser Met Thr Leu Mst I
Fnu4H Fnu4H
CTT ATA TCT GGT GGC GTA ACA CCT GCT GCA AAT GCT GCG CAA
Leu Ile Ser Gly Gly Val Thr Pro Ala Ala Asn Ala Ala Gln F.

CAC GAT GAA GCT CAA CAA AAT GCT TTT TAT CM GTG TTA AAT s 9 7 His Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn ' ~ ':

'~7 CHART B (~ag~ 4)-Bcl I
Sau3A
ATG CCT AAC TTA AAC GCT GAT CAA CGT AAT GGT TTT ATC CAA
Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Sau3A
AGC CTT AAA GAT GAT CCA AGC CAA AGT GCT AAC GTT TTA GGT ~ 31 Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly GAA GCT CAA AAA CTT AAT GAC TCT CAA GCT CCA AAA GCT GAT
Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Mst I Sau3A Hae II
GCG CAA CAA AAT AAG TTC AAC AAA GAT CAA CAA AGC GCC TTC
Ala Gln Gln Asn Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe TAT GAA ATC TTG AAC ATG CCT AAC TTA AAC GAG GAG CAA CGC ~o~
Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Sau3A
AAT GGT TTC ATT CAA AGT CTT AAA GAC GAT CCA AGC CAA AGC
Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser ACT AAC GTT TTA GGT GAA GCT AAA AAA TTA AAC GAA TCT CAA
Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln :
A

Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys Glu Gln Gln Asn ~ ~ :
:

.: . .:, :,: .

3'1 CHART B ~ e~

GCT TTC TAT GAA ATC TTG AAC ATG CCT AAC TTG AAC GAA GAA
Ala Phe Tyr Glu lle Leu Asn Met Pro Asn Leu Asn Glu Glu Hind III
C~ CGC AAT GGT TTC ATC CAA AGC TTA AAA GAT GAC CCA AGT 1017 Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser CAA AGT GCT AAC CTT TTA GCA GAA GCT AAA AAG TTA AAT GAA
Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu TCT CAA GCA CCG AAA GCT GAT AAC AAA TTC AAC AAA GAA C M
Ser Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Hind III
GAA GAA CAA CGC AAT GGT TTC ATC CAA AGC TTA AAA GAT GAC
Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Hae II
CCA AGC CAA AGC GCT AAC CTT TTA GCA GAA GCT AAA AAG CTA
Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys GAA CAA CAA AAT GCT TTC TAT GAA ATT TTA CAT TTA CCT AAC
Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn 4o CHART B (~agQ ~

TTA ACT GAA GAA CAA CGT AAC GGC TTC ATC CAA AGC CTT AAA
Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Sau3A

Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys AAG CTA AAC GAT GCT CAA GCA CCA AAA GAG GAA GAC AAC AAC
Lys Leu Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn AAG CCT GGT AAA GAA GAC GGC AAC AAA CCT GGT AAA GAA GAC
Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp GGC AAC M A CCT GGT AAA GAA GAC AAC AAA AAC CTT GGC AAA l5 2 Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys Asn Leu Gly Lys GAA GAC GGC AAC AAA CCT GGT AAA GAA GAC AAC AA~A AAA CCT
Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro S7 : S8 : :

GGC AAA GAA GAT GGC AAC AAA CCT GGT AAA GAA GAC GGC AAC
Gly Lys Glu Asp Gly Asn Lys~Pro Gly~Lys Glu Asp~Gly Asn :

: :
:

: . : . . : .

~L~

CHART B ~a~e~

AAG CCT GGT AAA GAA GAT GGC MC AAA CCT GGT AAA GAA GAT 16 1~ 7 Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Sll GGC AAC AAG CCT GGT AAA GAA GAT GGC AAC AAG CCT GGT AAA
Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Cla I
Taq I

Glu Asp Gly Asn Gly Val Ile A sp Asp Lys Leu Ser Asn Met pBR322 EcoRI
AGA ATT CTT GAA GAC GAA AGG GCC TCG TGA
Arg Ile Leu Glu Asp Glu Arg Ala Ser ***

.. .. .

~2~

CHA~T C~ G. ~g~-~
EcoRV
Sau3A Rsa I Taq I
GAT CTG ACC TAC GGT GTA CTG GCC C.AT ATC GAA GCG GAA GAC

Dde I
CTG GCG CGT GAA GCG TCG TTT GCT CAG GGA TTA CGC GCG ATG

ATT GGC GGT ATC TTA ACC GCA TCC TGA TTC TCT CTC TTT TTC

GGC GGG CTG GTG ATA ACT GTG CCC GCG TTT CAT ATC GTA ATT

Eco RI
TCT CTG TGC AM AAT TAT CCT TCC CGG CTT CGG AGA ATT CCC

Nde I
CCC A~ ATA TTC .`CT GTA GCC ATA TGT CAT GAG AGT TTA TCG

Taq I
TTC CCA ATA CGC TCG AAC GAA CGT TCG GTT GCT TAT TTT ATG

Hinc II Aha III Sau3A
GCT TCT GTC AAC GCT GTT TTA AAG ATT AAT GCG ATC TAT ATC

Sau3A
ACG CTG TGG GTA rTG CAG TTT TTG GTT TTT TGA TCG CGG TGT

-10 Nco I -------------CAG TTC TTT TTA TTT CCA TTT CTC TTC CAT GGG TTT CTC ACA

.:,, , ~ ~

.
-.

43 ~ 5~7 CHART C J~gC ~
Hinc II
---'----------------- Hpa I
GAT AAC TGT GTG CAA CAC AGA ATT GGT TAA CTA ATC AGA TTA

Hinc II RBS
AAG GTT GAC CAG TAT TAT TAT CTT AAT GAG GAG TCC CTT ATG
Met Taq I
TTA CGT CCT GTA GAA ACC CCA ACC CGT GAA ATC AAA AAA CTC
Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Mst I

Asp Gly Leu Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe protein A

Bcl I
Sau3A
TAT CAA GTG TTA AAT ATG CCT AAC TTA AAC GCT GAT CAA CGT
Tyr Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Sau3A
AAT GGT TTT ATC CAA AGC CTT AAA GAT GAT CCA AGC CAA AGT
Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser GCT AAC GTT TTA GGT GAA GCT CAA AAA CTT AAT GAC TCT CAA
Ala Asn Val Leu Gly Glu Ala~Cl~ Lys Leu Asn Asp 3er Gln :

:
`` , :

,. . , . ~ , ~

CHART C (~a~P 3 ) Mst I Sau3A
GCT CCA AAA GCT GAT GCG CAA CM AAT AAG TTC AAC AAA GAT
Ala Pro Lys Ala Asp Ala Gln Gln Asn Lys Phe Asn Lys Asp Hae II

Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu AAC GAG GAG CAA CGC AAT GGT TTC ATT CAA AGT CTT AAA GAC
Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp ;

Sau3A
GAT CCA AGC CAA AGC ACT AAC GTT TTA GGT GAA GCT AAA AAA
Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn AAA GAA CAA CAA AAT GCT TTC TAT GAA ATC TTG AAC ATG CCT
Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Hind III
AAC TTG AAC GAA GAA CAA CGC AAT GGT TTC ATC CAA AGG TTA
Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu AAA GAT GAC CCA AGT CAA AGT GCT AAC CTT TTA GCA GAA GCT s 4 9 Lys Asp Asp Pro S~r Gln Ser Al~ Asn Leu Leu Ala Glu Ala ~ ~ "
.
: ~

, i5~ 7 CHART C ~ r ~ ge h?

AAA AAG TTA AAT GAA TCT CAA GCA CCG AAA GCT GAT AAC AAA
Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Hind III
TTA CCT AAC TTA AAT GAA GAA CAA CGC AAT GGT TTC ATC CAA
Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Hae II
AGC TTA AAA GAT GAC CCA AGC CAA AGC GCT AAC CTT TTA GCA
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp AAC AAA TTC AAC AAA GAA CAA CAA AAT GCT TTC TAT GAA ATT
Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile TTA CAT TTA CCT AAC TTA ACT GAA GAA CAA CGT AAC GGC TTC
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Sau3A
ATC CAA AGC CTT AAA GAC GAT CCT TCA GTG AGC AAA GAA ATT 8 ~ 5 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile TTA GCA GAA GCT AAA AAG CTA:AAC GAT GCT CAA GCA CCA AAA
Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys `

.. , ., ~ ... .. : ,~ . : ,.. . .
,, ~

,~ - - : . . , ~ ,. ..
, . : .

CHART C-~g~5-~

Sl S2 GAG GAA GAC AAC AAC AAG CCT GGT AAA GAA GAC GGC AAC AAA
Glu Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys , .
CCT GGT AAA GAA GAC GGC AAC AAA CCT GGT AAA GAA GAC AAC l o Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn AAA AAC CTT GGC AAA GAA GAC GGC AAC AAA CCT GGT AAA GAA
Lys Asn Leu Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu GAC AAC AAA AAA CCT GGC. AAA GAA GAT GGC AAC AAA CCT GGT
Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp (,ly Asn Lys S10 Sll CCT GGT AAA GAA GAT GGC AAC AAG CCT GGT AAA GAA GAT GGC
Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly .

- .. .,. : .

., . ..:
::

~ 2~ 77 CHART C ~p~gP 6) Cla I
Taq I

Asn Lys Pro Gly Lys Glu Asp Gly Asn Gly Val I le ~ sp Asp pBR322 EeoRI
AAG CTG TCA AAC ATG AGA ATT CTT GAA GAC GAA AGG GCC TCG
Lys Leu Ser Asn Met Arg Ile Leu Glu Asp Glu Arg Ala Ser TGA
,~**

~ ~' ' :: :

'-f 'l 7>7 CT.-~Rl' D--p~G3-2 ~a~
EcoRV
Sau3A Rsa I Taq I
GAT CTG ACC TAC GGT GTA CTG GCC GAT ATC GAA GCG GAA GAC

Dde I
CTG GCG CGT GAA GCG TCG TTT GCT CAG GGA TTA CGC GCG ATG

ATT GGC GGT ATC i'TA ACC GCA TCC TGA TTC TCT CTC TTT TTC

GGC GGG CTG GTG ATA ACT GTG CCC GCG TTT CAT ATC GTA ATT

Eco RI
TCT CTG TGC AAA AAT TAT CCT TCC CGG CTT CGG AGA ATT CCC

Nde I
CCC AAA ATA TTC ACT GTA GCC ATA T~T CAT GAG AC-T TTA TCG

-Taq I
TTC CCA ATA CGC rc G AAC GAA CGT TCG GTT GCT TAT TTT ATG

Hinc II Aha III ` Sau3A
GCT TCT GTC AAC GCT GTT TTA AAG ATT AAT GCG ATC TAT ATC

Sau3A
ACG CTG TGG GTA TTG CAG TTT TTG GTT TTT TGA TCG CGG TGT

-10 Nco I -------------CAG TTC TTT TTA TTT CCA TTT CTC T~C CAT GGG TTT CTC ACA

. ~ - : . . . .

, .

~Cl )77 CHART D (p~sc ~
Hinc II
---`----------------- Hpa I
GAT AAC TGT GTG CAA CAC AGA ATT GGT TAA CTA ATC AGA TTA

Hinc II RBS
.~G GTT GAC CAG TAT TAT TAT CTT AAT GAG GAG TCC CTT ATG
Met Taq I
TTA CGT CCT GTA GAA ACC CCA ACC CGT GAA ATC AAA AAA CTC
Leu Arg Pro Val Glu Thr Pro Thr Arg Glu Ile Lys Lys Leu Mst I
GAC GGC CTT GCG CAA CAC GAT GAA GCT CAA CAA AAT GCT TTT 8' Asp Gly Leu Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe Drotein A

Bcl I
Sau3A
TAT CAA GTG TTA AAT ATG CCT AAC TTA AAC GCT GAT CAA CGT
Tyr Gln Vai Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Sau3A

Asn Gly Phe Ile Gln Ser Leu Lys Asp As? Pro Ser Gln Ser GCT AAC GTT TTA ~GGT GAA GCT CAA AAA CTT AAT GAC TCT CAA
Ala Asn Val Leu Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln .

, .
.

~5~7~

CHART D ~ge-~
D

' Mst I Sau3A
&CT CCA AAA GCT GAT GCG CAA CAA AAT AAG TTC AAC AAA GAT
Ala Pro Lys Ala ~sp Ala Gln Gln Asn Lys Phe Asn Lys Asp Hae II

Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu AAC GAG GAG CAA CGC AAT GGT TTC ATT CAA AGT CTT AAA GAC
Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Sau3A
&AT CCA AGC CAA AGC ACT AAC GTT TTA GGT GAA GCT AAA AAA
Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn A~ GAA C M CAA AAT GCT TTC TAT GAA ATC TTG AAC ATG CCT
Lys Glu Gln Gln Asn Ala Phe Tyr ;Glu lle Leu Asn Met Pro Hind III
AAC TTG AAC GM GAA CAA CGC AAT GGT TTC ATC C M AGC TTA
Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu AAA GAT GAC CCA ~GT CAA AGT GCT AAC CTT TTA GCA GAA GCT 5~,9 Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala :- ~ , iS~)~7 CHART D 1~gP ll ) B

AAA AAG TTA MT GM TCT CM GCA CCG AAA GCT GAT MC AAA
Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Hind III
TTA CCT AAC TTA MT GAA GAA CM CGC AAT GGT TTC ATC CAA
Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Hae I I
AGC TTA AAA GAT GAC CCA AGC CAA AGC GCT AAC CTT TTA GCA
Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala GAA GCT AM AAG CTA AAT GAT GCA CAA GCA CCA AAA GCT GAC 7 s 9 Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp AAC MA TTC MC AAA GM CM CAA MT GCT TTC TAT GAA ATT
Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile TTA CAT TTA CCT AAC TTA ACT GAA ~ GAA CAA CGT AAC GGC TTC
Leu His Leu Pro Asn Leu Thr Glu Glu Gln Ar~s Asn Gly Phe Sau3A
ATC CAA AGC CTT AAA GAC GAT CCT TCA GTG AGC :AAA GAA ATT 8 ~ 5 Ile Gln Ser Leu Lys Asp Asp Pro Ser Val Ser Lys Glu Ile .
TTA GCA GM GCT AAA AAG CTA AAC GAT GCT CAA GCA CCA AAA
Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys :

~ ~i5~ 7 CHART D (~ag~5 Sl GAG GAA GAC AAC AAC AAG CCT GGT AAA GAA GAC GGC AAC AAA
Glu Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys CCT GGT AAA GAA GAC GGC AAC AAA CCT GGT AAA GAA GAC AAC l o Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn AAA AAC CTT GGC AAA GAA GAC GGC AAC AAA CCT GGT AAA GAA
Lys Asn Leu Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu GAC AAC AAA AAA CCT GGC AAA GAA GAT GGC AAC AAA CCT GGT
Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys S10 Sll CCT GGT AAA GAA GAT GGC AAC AAG CCT GGT AAA GAA GAT GGC
Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly :

~3 7 ~

CHART D (-page 6) , stop AAC AAG CCT GGT AAA GAA GAC GGC AAC GGA GTC ATC GGG CGC
Asn Lys Pro Gly Lys Glu Asp Gly Asn Gly Val Ile Gly Arg linker GCT AGC TAG CTA GCG CGC CCG
Ala Ser '';`~* Leu Ala Arg Pro :

Claims (39)

1. A hybrid protein having the following amino acid sequence:

2. A hybrid protein having the following amino acid sequence:

3. A hybrid protein having the following amino acid sequence:

4. A recombinant DNA transfer vector comprising DNA having the following nucleotide sequence or equiva-lent nucleotide sequences containing bases whose trans-lated region codes for the same amino acid sequence:

5. A recombinant DNA transfer vector comprising DNA having the following nucleotide sequence or equiva-lent nucleotide sequences containing bases whose trans-lated region codes for the same amino acid sequence:

6. A recombinant DNA transfer vector comprising DNA having the following nucleotide sequence or equiva-lent nucleotide sequences containing bases whose trans-laced region codes for the same amino acid sequence:

7. The DNA transfer vector of claim 4 transferred to and replicated in a prokaryotic microorganism.

8. The DNA transfer vector of claim 7 wherein said prokaryotic microorganism is an E. coli K-12 derivative.

9. The DNA transfer vector of claim 5 transferred to and replicated in a prokaryotic microorganism.

10. The DNA transfer vector of claim 9 wherein said prokaryotic microorganism is an E. coli K-12 derivative.

11. The DNA transfer vector of claim 6 transferred to and replicated in a prokaryotic microorganism.

12. The DNA transfer vector of claim 11 wherein said prokaryotic microorganism is an E. coli K-12 derivative.

13. Plasmid pBG9 as shown in FIGURE 2 of the drawings.

14. Plasmid pBG5 as shown in FIGURE 3 of the drawings.

15. Plasmid pBG3-2 as shown in FIGURE 4 of the drawings.

16. Plasmid pBG3-2.DELTA.N as shown in FIGURE 5 of the drawings.

17. A microorganism transformed by the transfer vector of claim 4.

18. A microorganism transformed by the transfer vector of claim 5.

19. A microorganism transformed by the transfer vector of claim 6.

20. E. coli PR13(pBG9), a microorganism according to claim 17.

21. E. coli PR13(pBG5), a microorganism according to claim 18.

22. E. coli PR13(pBG3-2), a microorganism accord-ing to claim 19.

23. E. coli PR13(pBG3-2.DELTA.N).

24. A process for preparing recombinant plasmid pBG9 which comprises (a) cutting plasmid pBG101-41 with endonuclease BamHI and blunting by treatment with Bal-31 exonuclease;
(b) cutting pBG101-41 further with ClaI at the unique site in the pBR322 DNA;
(c) obtaining a blunt-ClaI protein A fragment from plasmid pAc37, and (d) coupling the construction of (c) with (b) to obtain plasmid pBG9.

25. A process for preparing recombinant plasmids pBG3-2 and pBG3-2.DELTA.N which comprises (a) digesting plasmid pBR325 with ClaI and SalI
and isolating the 5368 bp fragment;
(b) digesting pBG5 with ClaI and SalI and isolating the 2000 bp fragment;
(c) ligating the fragments obtained in (a) and (b);
(d) digesting the ligated product of (c) with ClaI and isolating a linear molecule of 7.4 kb;
(e) ligating said 7.4 kb molecule with a linker DNA fragment containing stop codons to obtain plasmid pBG3-2;
(f) digesting plasmid pBG3-2 with restriction endonuclease Nde;
(g) extracting said digested plasmid with phenol-ether and precipitating with ethanol; and (h) religating said product DNA obtained in (g) at dilute DNA concentration to obtain plasmid pBG3-2.DELTA.N.

26. A process for preparing a hybrid protein having the following amino acid sequence:

which comprises culturing a prokaryotic microbe hosting a recombinant DNA transfer vector comprising DNA having the following nucleotide sequence or equivalent nucleo-tide sequences containing bases whose translated region codes for the same amino acid sequence:

27. A process, according to claim 26, wherein said prokaryotic microbe is an E. coli K-12 derivative with a lon or pnp mutation and said recombinant DNA
transfer vector is plasmid pBG9.

28. A process, according to claim 27, wherein said E. coli K-12 derivative is E. coli SG20251 or E. coli PR13.

29. A process for preparing a hybrid protein having the following amino acid sequence which comprises culturing a prokaryotic microbe hosting a recombinant DNA transfer vector comprising DNA having the following nucleotide sequence or equivalent nucleo-tide sequences containing bases whose translated region codes for the same amino acid sequence:

30. A process, according to claim 29, wherein said prokaryotic microbe is an E. coli K-12 derivative with a lon or pnp mutation and said recombinant DNA transfer vector is plasmid pBG 5.

31. A process, according to claim 30, wherein said E. coli K-12 derivative is E. coli SG20251 or E. coli PR13.

32. A process for preparing a hybrid protein having the following amino acid sequence:

which comprises culturing a prokaryotic microbe hosting a recombinant DNA transfer vector comprising DNA having the following nucleotide sequence or equivalent nucleo-tide sequences containing bases whose translated region codes for the same amino acid sequence:

33. A process, according to claim 32, wherein said prokaryotic microbe is an E. coli K-12 derivative with a lon or pnp mutation and said recombinant DNA transfer vector is plasmid pBG3-2 or plasmid pBG3-2.DELTA.N.

34. A process, according to claim 33, wherein said E. coli K-12 derivative is E. coli SG20251 or E. coli PR13.

35. A process for preparing useful proteins which comprises culturing a prokaryotic microbe hosting a recombinant DNA transfer vector derived from the E. coli colEl plasmid comprising .beta.-glucuronidase gene DNA, said vector having the rop gene partially or totally deleted or otherwise inactivated.

36. A process, according to claim 35, wherein said.
.beta.-glucuronidase gene DNA is obtained from an E. coli K-12 derivative.

37. A process, according to claim 36, wherein said E. coli K-12 derivative is E. coli MS371.

38. A process, according to claim 35, wherein said prokaryotic microbe is an E. coli K-12 derivative with a lon or pnp mutation.

39. A process, according to claim 38, wherein said E. coli K-12 derivative is E. coli SG20251 or E. coli PR13.