CA2175590A1 - Method of producing recombinant dimeric enzyme - Google Patents
Method of producing recombinant dimeric enzymeInfo
- Publication number
- CA2175590A1 CA2175590A1 CA 2175590 CA2175590A CA2175590A1 CA 2175590 A1 CA2175590 A1 CA 2175590A1 CA 2175590 CA2175590 CA 2175590 CA 2175590 A CA2175590 A CA 2175590A CA 2175590 A1 CA2175590 A1 CA 2175590A1
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- Prior art keywords
- enzyme
- dna
- leu
- subunit
- prokaryotic host
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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Abstract
This invention relates to a method of producing a recombinant eukaryotic heterodimeric enzyme using a prokaryotic host. The method involves constructing a first DNA vector containing DNA encoding one of the subunits of the dimeric emzyme and then constructing a second DNA vector containing DNA encoding the second subunit of the enzyme. Once the DNA vectors are constructed, they are used to transform a prokaryotic host. The transformed prokaryotic host cell is then cultured under conditions appropriate for the expression of the dimeric enzyme. For example, using the method of the present invention, the heterodimeric isoform of creating kinase CKMB can be produced. This invention further relates to a method of producing a recombinant human dimeric enzyme in an active form using a prokaryotic host, the recombinant enzyme products produced using the method of the present invention, and a transformed prokaryotic host constructed by the method of the present invention.
Description
~I Wo 9S112662 2 1 ~ 5 ~ 9 0 PC~IUS94/12624 MET}70D OF P~ODVCING r~7T
DIM16~IC 13N?Z YME
n~ 7. c~--n~ ~f 7-h~ Tnvf~n~ n The analysis of creatine kinase (CK) isoforms is important for the early diagnosis of acute myocardial 10 infarction (AMI) and for the early det~orminAtion of coronary artery reperfusion in patients treated with thrombolytic therapy (Alan and Wu, rlAhoratorv M~rl;c;ne, 23 (5) :297-302, 1992 ) . The CR isoforms CKI~M and CKMB can be used to determine the success of reperfusion therapy, although 5 meabuL. t of MB isoforms provides the earliest and most definitive results. Levels of ~ isoforms are also elevated in patients with skeletal muscle disease, and together with the relative MB index, can be useful for de~Prm;n;n~-S whether the muscle damage is acute or chronic.
Pure CK~IB is needed for research studies of myocardial metaholism and the enzyme ~ s catalytic ~ hAn; m and for preparation of standards and ~uality control materials for clinical analysis. Current methods of obtaining CK~B involve homogenlzing heart tissue and 25 precipitating the CKMB with ethanol or ammonium sulfate followed by ion exchange, gel filtration, and/or affinity chromatography (Grace and Roberts, ~~l;n ~~h~m ~-ta, 12~:59-71, 1982; and Herman and Roberts, ~nAl Bio~-h~m, 106:244-252, 1980) . These procedures involve multiple column-30 purification steps, are long and tedious, and may result in poor yields and low specific activities. In addition, the extracts of CI~MB can contain large amounts of contAm;nAntq, such as albumin which co-fractionates and co-migrates with C~MB on chromatography and pathogens re~uiring special 3 5 hAn(ll ;n--,, A need exists to improve and simplify the production of the different isoforms of CK to satisfy the rec,uirements of researchers and rl;ni~ 3nq W095112662 ; PCFIUS94/12624 ~
21~rj9~ - 2 -S v of~ th~a Invention This ;n~r~nt;n"~ relates to a method of producing a rP~ ' ,; nAnt eukaryotic heterodimeric enzyme in an active form usi~g a prokaryotic host. The method involves 5 constructing a first 3NA vector containing DNA encoding one of the subunits of the heterodimeric enzyme and, then constructing a second DNA vector c~mt~;n;n~ DNA encoding the second subunit of the enzyme. Once the DNA vectors are constructed they are used to transform a prokaryotic host.
10 The transformed prokaryotic host cell is then cultured under conditions appropriate for the~ ~xpression of the heterodimeric enzyme. For example, using the method of the E~resent invention, the heterodimeric ios:Eorm of creatine kinase, i.e., C~NB, can be produced.
I 5 This invention further relates a method oi producing a recombinant human dimeric enzyme in an active form using a prokaryotic host.
Furthermore, the present inYention relates to the rprnrrhi n=lnt enzyme products produced using the method of
DIM16~IC 13N?Z YME
n~ 7. c~--n~ ~f 7-h~ Tnvf~n~ n The analysis of creatine kinase (CK) isoforms is important for the early diagnosis of acute myocardial 10 infarction (AMI) and for the early det~orminAtion of coronary artery reperfusion in patients treated with thrombolytic therapy (Alan and Wu, rlAhoratorv M~rl;c;ne, 23 (5) :297-302, 1992 ) . The CR isoforms CKI~M and CKMB can be used to determine the success of reperfusion therapy, although 5 meabuL. t of MB isoforms provides the earliest and most definitive results. Levels of ~ isoforms are also elevated in patients with skeletal muscle disease, and together with the relative MB index, can be useful for de~Prm;n;n~-S whether the muscle damage is acute or chronic.
Pure CK~IB is needed for research studies of myocardial metaholism and the enzyme ~ s catalytic ~ hAn; m and for preparation of standards and ~uality control materials for clinical analysis. Current methods of obtaining CK~B involve homogenlzing heart tissue and 25 precipitating the CKMB with ethanol or ammonium sulfate followed by ion exchange, gel filtration, and/or affinity chromatography (Grace and Roberts, ~~l;n ~~h~m ~-ta, 12~:59-71, 1982; and Herman and Roberts, ~nAl Bio~-h~m, 106:244-252, 1980) . These procedures involve multiple column-30 purification steps, are long and tedious, and may result in poor yields and low specific activities. In addition, the extracts of CI~MB can contain large amounts of contAm;nAntq, such as albumin which co-fractionates and co-migrates with C~MB on chromatography and pathogens re~uiring special 3 5 hAn(ll ;n--,, A need exists to improve and simplify the production of the different isoforms of CK to satisfy the rec,uirements of researchers and rl;ni~ 3nq W095112662 ; PCFIUS94/12624 ~
21~rj9~ - 2 -S v of~ th~a Invention This ;n~r~nt;n"~ relates to a method of producing a rP~ ' ,; nAnt eukaryotic heterodimeric enzyme in an active form usi~g a prokaryotic host. The method involves 5 constructing a first 3NA vector containing DNA encoding one of the subunits of the heterodimeric enzyme and, then constructing a second DNA vector c~mt~;n;n~ DNA encoding the second subunit of the enzyme. Once the DNA vectors are constructed they are used to transform a prokaryotic host.
10 The transformed prokaryotic host cell is then cultured under conditions appropriate for the~ ~xpression of the heterodimeric enzyme. For example, using the method of the E~resent invention, the heterodimeric ios:Eorm of creatine kinase, i.e., C~NB, can be produced.
I 5 This invention further relates a method oi producing a recombinant human dimeric enzyme in an active form using a prokaryotic host.
Furthermore, the present inYention relates to the rprnrrhi n=lnt enzyme products produced using the method of
2 0 the present invention .
The invention further relates to a transformed prokaryotic host constructed by the method of the present invention .
2 5 Brief Descril~tion of tllf' Drawina Figure 1 a schematic illustration of the method of producing a rpr~mhinr~nt dimeric enzyme of the present invention .
The invention further relates to a transformed prokaryotic host constructed by the method of the present invention .
2 5 Brief Descril~tion of tllf' Drawina Figure 1 a schematic illustration of the method of producing a rpr~mhinr~nt dimeric enzyme of the present invention .
3 0 I~n; ~ scr;ntion of the Inventio~
This invention is based upon the discovery that ;ffP~Gnt isoforms of creatine kinase ~i.e., C~'~BB, CK[~I and C~MB) can be produced by constructing a DNA vector for the two different subunits of the enzyme and tr~ncfrn~;n~ a host 35 cell with the two DNA vectors, the resulting transformed host cell being capable of expressing CKBB, CKM~I and CKMB.
DNA v~ctor The term "DNA vector~' is intended any
This invention is based upon the discovery that ;ffP~Gnt isoforms of creatine kinase ~i.e., C~'~BB, CK[~I and C~MB) can be produced by constructing a DNA vector for the two different subunits of the enzyme and tr~ncfrn~;n~ a host 35 cell with the two DNA vectors, the resulting transformed host cell being capable of expressing CKBB, CKM~I and CKMB.
DNA v~ctor The term "DNA vector~' is intended any
4 0 replication competent vector which has the capability of having a DNA fragment inserted into it and, subse~uently, the expression of that DNA insert by an appropriate host W095/~2662 2 1 7 ~ 5 3 ~ PCFrUS94/12624 cell. In addition, the DNA vector must be receptive to t~e insertion of a DNA fragment containing the DNA where the sec7uence encodes the subunits of the target eukaryotic dimeric enzyme such as creatine kinase ~i.e., Cl~M and CRB).
S E'urth. - e, the DNA vector must contain a promoter which can be recognized by the host cell . Procedures f or the construction of DNA vectors include those described in MAniAtiC et al., Moler111Ar Clon;n~, A LAhr~ratrrv MAnllAl, 2d, Cold Spring Harbor Laboratory Press (1989), herein referred 0 to as Maniatis et al..
The term ~DNA fragment" is intended to Pnr~ ~qs any DNA fragment that encodes an enzyme subunit.
The DNA ragment once inserted into a DNA vector should be transmittable to a host microorganism by transformation or 15 conjugation or transfection. Procedures for the construction or extraction of DNA fragments include those described in Maniatis et al, and others known by those skilled in the art.
2 0 Host The trans~ormed prokaryotic host of the present invention can be created by various methods by those skilled in the art. For example, transfection, trancformation or el~,LL~ ,Lcltion as explained by Maniatis et al. can be used.
2 5 By the term ~prokaryotic ~ost" is intended any prokaryote capable of the uptake and expression of foreign DNA, i . e., DNA not originally a part of the prokaryotes ~ s nuclear material. Suitable prokaryotes may include Corynebacterium, Escherichia, Stre~tacyces or RAri 7 7~,c, 7~ t Dlm~r i c 7!:~zy~n~
The rP~ ~; nAnt dimeric enzyme of the present invention is int~n~lP~ to rn~ _ .C~ any protein consisting of two subunits and possessing enzymatic properties.
3 5 The invention will be further illustrated by the fol 1 owing non-limiting 7~YPmrl i ~ication:
WO 9~112662 , PC~IUS94/12624 ~
2 1 ~
~lr~MPLIFICA~ION
MatQrials and ~thods:
Clonin~ (`KMR .-r)N~ llqin~ PCR
DNA f ragments carrying cDNA encoding CKM and
S E'urth. - e, the DNA vector must contain a promoter which can be recognized by the host cell . Procedures f or the construction of DNA vectors include those described in MAniAtiC et al., Moler111Ar Clon;n~, A LAhr~ratrrv MAnllAl, 2d, Cold Spring Harbor Laboratory Press (1989), herein referred 0 to as Maniatis et al..
The term ~DNA fragment" is intended to Pnr~ ~qs any DNA fragment that encodes an enzyme subunit.
The DNA ragment once inserted into a DNA vector should be transmittable to a host microorganism by transformation or 15 conjugation or transfection. Procedures for the construction or extraction of DNA fragments include those described in Maniatis et al, and others known by those skilled in the art.
2 0 Host The trans~ormed prokaryotic host of the present invention can be created by various methods by those skilled in the art. For example, transfection, trancformation or el~,LL~ ,Lcltion as explained by Maniatis et al. can be used.
2 5 By the term ~prokaryotic ~ost" is intended any prokaryote capable of the uptake and expression of foreign DNA, i . e., DNA not originally a part of the prokaryotes ~ s nuclear material. Suitable prokaryotes may include Corynebacterium, Escherichia, Stre~tacyces or RAri 7 7~,c, 7~ t Dlm~r i c 7!:~zy~n~
The rP~ ~; nAnt dimeric enzyme of the present invention is int~n~lP~ to rn~ _ .C~ any protein consisting of two subunits and possessing enzymatic properties.
3 5 The invention will be further illustrated by the fol 1 owing non-limiting 7~YPmrl i ~ication:
WO 9~112662 , PC~IUS94/12624 ~
2 1 ~
~lr~MPLIFICA~ION
MatQrials and ~thods:
Clonin~ (`KMR .-r)N~ llqin~ PCR
DNA f ragments carrying cDNA encoding CKM and
5 CKB proteins that had been cloned from a human cDNA library have been descr~bed (Perryman et al, Bio~llP~ . ~In~ l Biol~hvs .
E~P:~qP;Ircl~ C .. 140: 981-989, 1986; and Villarreal-Levy et al ., Bi~ Pnl . ~n~l Bio~hvs . Reasearch Comm. . 144 :1116-1127, 1987~ . We used PCR ampl;fi~=t; ~n to change the DNA sequence 10 at the N- and C- termini t~ add restriction en~yme sites that were suitable for cloning the CK cDNA into Genzyme expression vectors . PCR primers were syntl~Pq; 7.P~l at Genzyme.
The primers had the following 5 - 3 sequences;
sL22 (Ndel site at the ATG start codon of CKB) GCC CAT ATG
CCC TTC TCC A~C AGC CAC A
SL23 (Eco~1 site after the stop codon of CKB~ GGA ATT CAT
TTC TGG GCA GGC ATG AGG
SL2~ (Ndel site at the ATG start codon of CKM) GCC CAT ATG
CCA TTC GGT AAC ACC CAC AAC
20 SL25 (Bam~1 site after the stop codon of C}~M) GCA GGA TCC
TAC TTC TGG GCG GGG ~TC AT.
The GeneAmp PCR Reagent ~it with AmpliTaq DNA
Polymerase from Perkin Elmer Cetus (Norwalk, CT) was used for PCR reactions. The reactions were carried out following 2 5 standard procedures outlined in the literature enclosed in the kit Specifically, 5-30 ng DNA, 100 pmol primer DNA, 2.5 U AmpliTaq DNA polvmerase, and 200 ,umol dATP, dCTP, dGTP, dTTP were mixed with supplied buffer, and the reaction mix was overlayed with Ampliwax ( Perkin Elmer Cetus ) . The 3 0 PCR machine (Coy Laboratory Products, Inc . Grass Lake, MI) was ~L yL -d for ~ cycle of 94-C Lmelt) for 2 minutes, 55 C (anneal) for 2 minutes, 72 C (extend~ for 2 minutes, this cycle was repeated 1~3 times. A final extension step was run for 10 minutes to allow for complete polymerization 3 5 of all strands. PCR product (approximately 5-10 llg~ was digested with Ndel and EcoR1 ~for CKB~ or Ndel and BamH1 ~ for CKM) and purified by electrophoresis through a 0.7% low melting point agarose TAE buffered gel (FMC BioProducts, Inc. Rockland, ME~ for cloning into expression vectors.
40 Electrophoresis was performed as described in Molecular Cloning (Sambrook, Fritsch and M~n;~ti c, 198g. Cold Spring Harbor Press, Cold Spring Har~or, NY) . Restriction WO 95112662 217 rj S ~l ~ PC~IUS94/12624 endonucleases were purchased from New England BioLabs (Beverly, MA) and digestion reactions set up as suggested by the manufacturer. DNA fragments were purified from gel slices using the f.~nf~ An kit (BiolO1, La Jolla, CA~.
s Conqtruct;n~ thf~ S;~n - ~rnreccinn Vector r-R7~8 The expression vector was constructed at Genzyme as a derivative of the plasmid pBluescript SK +/-~available from Stratagene, La Jolla, CA). Expression is 10 driven off the Lac promoter. Our vector, pRZ38, was constructed by adding an restriction enzyme site at the ATG
start codon of the b-galactosidase gene through site-directed mutagenesis. Mutagenesis protocols were followed as described using the Muta-Gene In Vitro 15 Mutagenesis Rit from BioRad (Ri rl 1 , CA) . This change in the vector allows cloning of foreign genes after the LacZ
promoter but maintains similar spacing from the promoter as in the native gene.
20 ('lnn;n~t CKM ein~ CKR nN!~ ;nt~ R7~8 Vector DNA (5 llg) was digested with Ndel and EcoR1 (for CKB) or Ndel and BamH1 (for CKM~ and gel purified as above. Approximately 100 ng digested vector DNA and 100 ng digested PCR product were ligated in a 20 111 reaction (T4 25 DNA ligase purchased from NEB, Beverly, MA) . Ligation reactions set up as described in Molecular Cloning. After overnight ;n llh~t;nn at 15 C ligation mixes were diluted to 60~1 with dlI20. 1111 of each was electroporated into electroporation competent E. coli strain MC1061. The 3 0 CeLl-Porator and Voltage Booster were purchased from Bethesda Research Labs (Bethesda, MD~ . Protocols or competent cell preparation and ele.LL~.~ v, ltion are described in the I~struction manual. Transformants were selected on-LB agar plates supplemented with 50 ,~Lg~ml ampicillin.
3 5 Transformants were analyzed for harboring the correct recombinant plasmid using the alkaline lysis miniprep t~ hn;qr1c. (Molecular Cloning) . Miniprep DNA was analyzed by restriction enzyme mapping. 25 ml cultures of pRZ52 (CKB) and pRZ53 (C~¢) were grown in LB with 5011g/ml ampicillin and 40 larger scale DNA preps were purified using QIAGEN plasmid purification columns (QIAGEN Corp., Chatsworth, CA) .
Dideoxynucleoside chain-tF~rm;nAt;nn DNA secluencing reactions WO 95112662 ~ PC~IUS94112624 2~7~a were carried out according to the standard protocols described in the Sequenase 2 O kit from USB tCleveland, OH).
[-35S]-dATP (~ew ~ngland Nuclear~ was used to radiolabel the sequences for visualization on Kodak XAR film. Reactions 5 were separated through a 6% polyacrylamide gel, the gel dried, and expbsed to ilm as described in Molecular Cloning. The DNA sequence representing the coding regions of CKs and CKM are in ~igure G1 and 2.
Once the CKM DNA sequence was ~lPtPrmin~9 to be 10 correct the a~npicillin resistance gene in pRZ53 was exchanged for the kanamycin resistance gene from Tn903 ~Nomura et al., ~ene, 3:39-51, 1987). The ampicillin gene was cut out using the restriction enzymes Ssp~ and spml, and the vector ends were blunt ended using T4 polymerase. The 15 kanamycin resistance gene had been cloned into the polylinker in a pBR322 :vector. The gene was cut out of the vector using BamH1 and was blunt ended with T4 polymerase.
The resulting C~M plasmid is referred to as pRZ69.
20 t`orc:truct; n~ the CRMl~ Co-P~ression Strain pRZ52 and pRZ69 DNA was mixed together in a concentration o~ approximately 50 llg/ml and 1 1ll of the mixture electroporated into 20 1ll MClû61 cells. The cells were plated onto 1~3 plates containing 50 llg/ml ampicillin 25 and 50 llg/ml kanamycin to select or cells that had been co-tra~sformed with both plasmids. Co-transformants were analyzed ~y restriction digestion of miniprep DNA (the standard tP~ n; q~P is described in Molecular Cloning) .
WO 95112662 ~ 1 7 5 5 9 0 pC~ S94/12624 p\nA 1 vr i n F~-~nressinn Expression analysis of the co-transformants was carried out as follows. Clones were grown overnight in 2 ml LB, 0.2~s glucose, 50 llg/ml kanamycin and 50 llg/ml S i ~71l;n at 37 C shaking. In the morning 25 ml of LB with 50 ~Lg/ml kanamycin and 50 llg/ml ampicillin was inoculated with 0 . 6 ml of the overnight culture and grown at 30 C
shaking. Cultures were grown to A600 about 0.4 then sampled at one hour intervals for 4 hours. Cultures were left 10 growing overnight and an additional sample taken. Whole cell samples were boiled in SDS-PAGE sample buffer and run through 12% polyacrylamide gels to assess protein production Gels were obtained f rom BioRad, and protocols provided with gels were followed.
Several assays were used to ~F.t~rmin~ the level and quality of the expressed r~ mhinAnt proteir,. Cultures were grown as previously described. At A600 approximately 1. 0 or af ter overnight incubation cells were harvested and resuspended in a lysis buffer (20 mM Bis-Tris pH 6.9, 0.259 20 Tween 20, 10 mM b ~ptoethanol, 10 mM EDTA, 10 mM EGTA, lmM PNSF). The resuspended cells were lysed by sonication on ice (or for larger scale analysis cells were lysed using the Microfl~ ;7~r from Microfluidics Corp., Newton, MA ) and the cell debris removed by centrifugation. Samples were 2 5 analyzed using the following assay systems after diluting into lysis buffer. The Creatine Kinase Reagent (Sigma 47-UV) is a spectrophotometric assay for kinetic determination of enzyme activity. The Creatine Phosphokinase (CPK) Isoenzymes Kit (Sigma 715-EP) separates 3 0 the various isoforms (NM, MB, and BB) and stains for activity. The CR-MB assay system for the Abbott ~c analyzer uses a microparticle enzyme i oAqsay (MEIA) to determine specific protein mass of CR-MB in a sample. (A11 protocols are provided with the assay kits.
:~uiv)- 1 ~nt ~
Those skilled in the art will recognize, or be able to ascertain, using no more than routine expGr;~ tAt;nn many equivalents to the specific embodiments 4 0 of the invention described herein. Such equivalents are ;nt.~n~ l to be ~- cced by the fallowing claims:
~O95/12662 j ' ~ PC~IUS94/12624 ~
2 ~ 8 -SEQUENCE LISTING
( l ) GENE-RAL INFORMATION:
(i~ APPLICANT: Ziegler, Robin ~.
Lon~, Sue ( ii ) TITLE OF LNVI~ I'l'LUN: Method of Producing Rl ' 'nAnt Dimeric Enzyme l O
(iii) NU~3ER OF ~U N(.~ : 4 ( iV) ~:U~ UNJ~;N~:~; ADDRESS:
(A~ ~nnR~. : Bill Gosz , Esq., 1 5 Genzyme Corporation (B) STREET: One Kendall Sc~uare ( C ~ CITY: Cambridge ( D ) STATE: MA
( E ) COUNTRY: U . S . A .
2 0 (F) ZIP: 02139 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) CC~PUTER: 1:~ PC compatible 2 5 (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l . 0, Version # l . 25 (vi) CURRENT APPLICATION DATA:
3 0 (A) APPLICATI~ NOMBER: US
( ~3 ) F ILING DATE:
( C ) CLAS S IF I CAT ION:
(viii) ATTORNEY/AGENT INFOR~TION:
3 5 (A) NAME: William G Go8z (B) REGISTRATION NOMBER: 27,787 (C) ~ ~/DOCKET NOMBER: GEN3-l0.0 WO 95/126G2 217 ~ ~ ~ O PC~/IIS941~2624 _ 9 _ ( ix) ~r~r ~ [ ~ ~ IN I ~ L l'ION INFORMATION
(A) ~rT T,T PT~nNF (617) 252--7583 (B) TELEFAX (617) 252-7600 ( C ) TELEX 2 012 23 GENCA~B
( 2 ) INFORMATION FOR SEQ ID NO
(i) SEQ~ENCE CHaRACTERISTICS
(A) LENGTH 11~6 base pairs (B) TYPE nucleic acid (C) S'rR~NlJ~l~N~S`i single (D) TOPOLOGY linear ( ii ) MnT F~'TTT~T. TYPE cDNA
(iii) ~YL~r~ L~lCAL NO
( iv) ANTI-SENSE YES
2 0 (v) FRAGMENT TYPE N-terminal (vi~ ORIGINAL SOURCE
(A) OR~ANISM Homo sapiens 2 5 ( xi ) SEQUENOE L~ ClL~ l lON SEQ ID NO l ATGCCCTTCT rr~ArAr.rrA CAACGCL~CTG AAr~rlrr~rr~rT L.~ W~ GGACGAGTTC 60 CCCGACCTGA r,rrrrrDrAA rAArrArATr~ GCCAAGGTGC ~rr.ArrrrrrA GCTGTACGCG 120 r-Arr-~Grr-rr~ rrAArAr~rAr r.rrrArrr~r TT,CACGCq'GG ACGACGTCAT rrAnArArrr 180 GTGGACAACC rr~r~rrArrr r.~rDrA~rA~r~ ACCGTGGGCT ~.1'1.1~:13 I~I.(j rr.Arr~Arr.Ar. 240 3 5 TCCTACGAAG TGTTCL~AGGA TCTCTTCGAC CCCATCATCG Ar~Arrrr.rA rrrr.rrir~rAr 300 - AAr.rrrArrn ATGACGACAA rArrrArrrr AArrrrr.ArA Arr~rrArr~ rrrrr.Arr.Ar 360 CTGGACCCC~ Ar~Arr~Tr~rT GAGcTcGr-GG GTGGCCACGG r~rrrrArrAT ~L~i~L . 4ao i~.~LLC: rrr~r~r.rAr. rrrrrrr~An rr,rrr.AnrrA TCGAGAAGCT rrrrr~rrrAA 480 WO 95/12662 . ~ PC'FIUS9411262-1 ~
2 17 ~ 0 r~ rrrTrr-Drrr rr.ArrTrr.rr. GGCCGATACT ACGCGCTCAA GAGCATGACG ~40 ~:Arrirrr.~r.r ArrDr~rDr~rT DTrr~ArnAr CACTTCCTCT Trr~DrAAr-rr ~ 600 5 ~l~ L~; rrTrr.r.f:rDT rrrrrrrr.Ar Tr~rrrr.Drr. rrr.rrrr.~AT rTrrrArDAT 660 r.ArDATAAr.A ~ l GTGGGTCAAC r.Arr.Arr.Arr ArrTrrr,r.~:T CATCTCCATG 720 r~r.AAr.rr~r7 rrAAr~Tr.AA GGAGGTGTTC ACCCGCTTCT r~rArrr~rrT CACCCAGATT 780 l O
GAAACTCTCT TrAAr.TrTAA GGACTATGAG TTCATGTGG,A ACCCTCDCCT GGGCTACATC 840 CTCACCTGCC CATCCAACCT rrr.rDrrr,r~ rTr,rrrr,r~r. GTGTCGATAT CAAGCTGCCC 900 nr~rDr~r~r~rG GTGTGGACAC ~l~ , TCGACGTCTC rD~rrr~TnAr 1020 ~l~iW-r TCTCAGAGGT r~nAnrTr~r~Tr~ CAGATGGTGG 'rr~GACGGAGT rADrrTrrTr 1080 ATCGAGATGG AACAGCGGCT rr.Drr~rrr.r CAGGCCATCG ACGACCTCAT r~rrTr~rrrAr~ 1140 AAATGA 119.6 ~IVO 95112662 2 1 7 5 ~ ~ ~ PC~IUS94l12624 1 ~ _ (2 ) INFORM~TION FOR SEQ ID NO: 2 ( i ) SEQ~ENCE CH~RACTERISTICS:
~A) LENGT~: 1146 base ~airs ~B) TYPE: nucleic acid ~ C ) S'T'T~ A N I I ~ 'i 'i: s irlgle ~D) ~OPOLOGY: linear ~ ii) M~T,T~`TTTT~' TYPE: c~A
l O
(iii) ~Ul'~ll~AL: NO
(iv) P~NTI-SENSE: YES
(v) FRAG~ENT TYPE: N-ter~ninal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ATGCCATTCG r.~AArArrrA CAACAAGTTC AAGCTGAATT ArAAnrrTnA rr.Arr.Ar.~Ar 60 CCCGACCTCA rrAA~rA~AA CAACCACATG r~rrAAr~r TGACCCTTGA ACTCTACAAG 120 AAGCTGCGGG ArAArr.~r.A~ CCCPLTCTGGC TTCACTGTA~ ACGATGTCAT rrAr.ArAr.r.A 180 2 5 GTGGACAACC CAGGTCACCC CTTCATCATG ACCGTGGGCT ~L~L~ TGATGAGGAG 240 TCCTPLCGAAG TTTTCAAGGA ACTCTTTGAC CCCATCATCT CGGATCGCCA rrrr~rr~Ar 3 o O
AAArrrAr~r. ArAArrAr~ GPCTGACCTC AArrA'rGAAA ACCTCAAGGG TGGAGACGAC 360 CTGGACCCCA Ar~rArr.~rr~ rAr.rArrrrr, GTCCGCPLCTG rrrrrArrAq~ rAAr~rr~rAr 420 ACGTTGCCCC CACACTGCTC CCrTGGCGAG ~ TGGAGAAGCT rTr~r.~r~AA 480 3 5 r.r~r~rrAArA GCCTGACGGG CGAGTTCAAA rrr.AAr.~Ar~ ACCCTCTGAA r.ArrA~r.Arr. 5~0 rAr.AArr.Arr AGCAGCAGCT CATCGATGAC CACTTCCAGT TCGACA~GCC WL~ U~ 600 ,~-~ CCTCAGGCAT rr,rrrrrrAr llr~r~rrrr~rr~ CCCCTGGCAT CTGGCACAAT 660 r.ArAAr~Ar.A ~7~lL~,l~il G~:GTGAAC GAGGAGGATC ACCTCCGGGT CATCTCCATG 720 W095112662 , PC~IUS9~112624 - 217~9a ~ 12-r~r.~r~rr~ GCAACATGAA CCZ~_LLLL~ - ~.~ I 1.~ 1 I~ I GCGTAGGGCT GCAGAAGATT 780 GAG~AGATCT TTAAGAAAGC TrrrrD.rrrr TTCI!TGTGaA ArrDrrArr~r GGGCTACGTG 840 Sr~rsrr~rrr cATccAAr-cT r.rrr~r~r.rr. r~rrnTrr.~r. GCGTGCATGT GAAGCTGGCG 900 r~rrTr~r~r~ ~r~rrrr~ GTTCGAGGAG ATCCTCACCC 4~Ll~i~4L~ rr~r~r~r. 960 rrl~r~rr.~r. CGGTGGACAC AGCTGCCGTG rrr~rrl~r.~rAT TTGACGTGTC CAACGCTGAT 1~20 CGTCCGAAGT Ar.~r~rrl~r. CAGCTGGTGG TGaATGGTGT GAAGCTCATG 1080 GTGC~AA.;~TGG AGAAGAAGTT rr.~r~ rr~r CAGTCCATCG ~rr.P~r~r~ rrrrnrrr~r. , 140 WO 95/12662 2 t 7 5 5 ~ O PC~IUS9*12624 ( 2 ) INFOKM~TION FOR SEQ ID NO: 3:
( i ) sEQr~ENcE rr~R ~
(A) LEWGTH: 381 amino acids (B) TYPE: arnino acid (C ) s~R~Nnr~r~NF ~.~: single (D) TOPOLOGY: linear (ii) Mr,r,r~(~rrr,r~ TYPE: protei~L
(v) FRAGMENT TYPE: N-terminal (xi) sEslrJENcE L~ KLL~L~ L~I: SEQ ID NO:3:
1 5 Iet Pro Phe Ser ~sn Ser His Asn Ala Leu Lys Leu Arg Phe Pro Al~
Glu Asp Glu Phe Pro A~P Leu Ser Ala His Asn Asn His ~et Ala Lys Val Leu Thr Pro Glu Leu Tyr Ala Asp Val Arg Ala Ly~ Ser Thr Pro Ser Gly Phe Thr Leu Asp Asp Val Ile Gln Thr Gly Val Asp Asn Pro 2 5 so ss 60 Gly His Pro Tyr Ile Yo~ Thr V~l Gly Cys V.ll Ala Gly Asp Glu Glu 6s 70 75 80 3 0 Ser Tyr Glu V~l Phe Lys Asp Leu Phe Asp Pro Ile Ile Glu Asp Arg His Arg Arg Tyr Lys Pro Ser AsP ASp Asp Lys Thr AsP Leu Asn Pro Asp Asn Leu Gln Gly Gly A~p Asp . Leu Asp Pro Asn Tyr Val Leu Ser 4 0 Ser Arg Val Ala Thr Gly Arg Ser Ile Arg Gly Phe Cys Leu Pro Pro WO 95/12C62 . ' ,` ' ` PCI!/US94112624~
- 21~9~ 14_ His Cys Ser Arg Gly Glu Arg Arq Ala Ile Glu Lys Leu Alo Val Glu l~S lS0 lSS 160 Ala Leu Ser Ser Leu Asp Gly Asp Leu Ala Gly Arg Tyr Tyr Ala Leu Lys Ser Met Thr Glu A1~ Glu Gln Gln Gln Leu Ile Asp Asp His Phe 0 Leu Phe Asp LYB Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala l9S 200 205 Arg Asp Trp Pro Asp Al~ A121 Ars~ Ile Trp His Asn Asp Asn Lys Thr Phe Leu Val Trp Val Asn Glu Glu Asp E~is Leu Arg Val Ile Ser Met Gln Lys Gly Gly Asn Met Lys Glu Val Phe Thr Arg Phe Cys Thr Gly Leu Thr Gln Ile Glu Thr Leu Phe Lys Ser Lys Asp Tyr Glu Phe Met 2 5 Trp Asn Pro His Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn Leu Gly Thr Gly Leu Arg Ala Gly Val Asp Ile Lys Leu l?ro Asn Leu Gly Lys His Glu Lys Phe Ser Glu Val Leu Lys Arg Leu Arg heu Gln Lys Arg Gly Thr Gly Gly Val Asp Thr Ala Ala Val Gly Gly Val Phe Asp Val Ser Asn Ala Asp Arg Leu Gly Phe Ser Glu Val Glu Leu Val Gln Met 4 0 Val V~l Asp Gly Val Lys Leu Leu Ile Glu Met Glu Gln Arg Leu Glu ~ WO 95112662 ~17 ~ 5 9 0 rc~Nsg4/12624 ~ 15 --Gln Gly Gln Ala Ile Asp Asp ~eu D~et Pro Al~ Gln Lys l O
~0 ~Vo 95/12662 , ~ ' , PCFIUS94112624 ~
21~S5~ - t6-( 2 ) rNFORMATION FOR SEQ m NO: 4:
( i ) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 381 a~ino acids /`
(B) TYPE: amino acid (C) Slr1?1~N~ N~:~;';: single (D) TOPOLO(~Y: linear (ii) MOT T~'TIT,T. TYPE: protein l O
(v) FR~T TYPE: N-terminal (xi ) SEQUENCE L~ ~L~L~16~N: SEQ ID NO: 4:
Met Pro Phe Gly Asn Thr His Asn Lys Phe Lys Leu Asn Tyr Lys Pro Glu Glu Glu Tyr Pro Asp Leu Ser Lys His Asn Asn His Met Al~ Lys Vel Leu Thr Leu Glu Leu Tyr Lys Lys Leu Arg Asp Lys Glu Ile Pro Ser Gly Phe Thr Vzll Asp Asp VP1 Ile Gln Thr Gly V~l Asp Asn Pro 2 5 so s5 60 Gly His Pro Phe Ile Met Thr Val Gly Cys Val Al~ Gly Asp Glu Glu 6s 70 75 80 3 0 ger Tyr Glu VP1 Phe Lys Glu Leu Phe Asp Pro Il~ l:le Ser Asp Arg His Gly Gly Tyr Lys Pro Thr Asp Lys His Lys Thr Asp Leu Asn His 100 105 ~ 110 Glu Asn Leu Lys Gly Gly Asp Asp Leu Asp Pro Asn Tyr V~l Leu Ser Ser Pro V- 1 Ar~ Thr Gly Arg Ser Ile ~ys Gly Tyr Thr Leu Pro Pro WO95112662 2~7~59D PC~IUS94112624 His Cys Ser Arg Gly Glu Arg Arg Ala Val Glu Lys Leu Ser Val Glu Ala Leu Asn Ser ~eu Thr Gly Glu Phe Lys Gly Lys Tyr Tyr Pro Leu Lys Ser Met Thr Glu Lys Glu Gln Gln Gln Leu Ile Asp Asp His Phe 0 Gln Phe Asp Lys Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala Arg His Trp Pro Asp Al~ Pro Gly Ile Trp His Asn Asp Asn Lys Ser l S
Phe Leu Val Trp Val Asn Glu Glu Asp His Leu Arg Val Ile Ser Met Glu Lys Gly Gly Asn Net Lys Glu Val Phe Arg Arg Phe Cys Val Gly 2 0 2~5 250 255 Leu Gln Lys Ile Glu Glu Ile Ph~ Lys Lys Ala Gly Hia Pro Phe Met 260 265 270 ~~
2 5 Trp Asn Gln His Leu Gly Tyr Val Leu Thr Cys Pro Ser Asn Leu Gly Thr Gly Leu Arg Gly Gly Val His Val Lys Leu Ala His Leu Ser Lys His Pro Ly~i Phe Glu Glu Ile Leu Thr Arg Leu Arçr Leu Gln Lys Arg Gly Thr Gly Ala Val Asp Thr Ala Ala Val Gly Ser Val Phe Asp Val er ~sn Ala Asp Arg Leu Gly Ser Ser Glu Val Glu Gln Val Gln Leu 4 0 Val Val Asp Gly Val Lys Leu Met Val Glu Met Glu Lys LYs Leu Glu WO 95/12662 , ~ PC~IUS94/;2624 21~5~a - 18 -Lys Gly Gln Ser Ile Asp Asp Met 'fle Pro ~1~ Gln Ly~
E~P:~qP;Ircl~ C .. 140: 981-989, 1986; and Villarreal-Levy et al ., Bi~ Pnl . ~n~l Bio~hvs . Reasearch Comm. . 144 :1116-1127, 1987~ . We used PCR ampl;fi~=t; ~n to change the DNA sequence 10 at the N- and C- termini t~ add restriction en~yme sites that were suitable for cloning the CK cDNA into Genzyme expression vectors . PCR primers were syntl~Pq; 7.P~l at Genzyme.
The primers had the following 5 - 3 sequences;
sL22 (Ndel site at the ATG start codon of CKB) GCC CAT ATG
CCC TTC TCC A~C AGC CAC A
SL23 (Eco~1 site after the stop codon of CKB~ GGA ATT CAT
TTC TGG GCA GGC ATG AGG
SL2~ (Ndel site at the ATG start codon of CKM) GCC CAT ATG
CCA TTC GGT AAC ACC CAC AAC
20 SL25 (Bam~1 site after the stop codon of C}~M) GCA GGA TCC
TAC TTC TGG GCG GGG ~TC AT.
The GeneAmp PCR Reagent ~it with AmpliTaq DNA
Polymerase from Perkin Elmer Cetus (Norwalk, CT) was used for PCR reactions. The reactions were carried out following 2 5 standard procedures outlined in the literature enclosed in the kit Specifically, 5-30 ng DNA, 100 pmol primer DNA, 2.5 U AmpliTaq DNA polvmerase, and 200 ,umol dATP, dCTP, dGTP, dTTP were mixed with supplied buffer, and the reaction mix was overlayed with Ampliwax ( Perkin Elmer Cetus ) . The 3 0 PCR machine (Coy Laboratory Products, Inc . Grass Lake, MI) was ~L yL -d for ~ cycle of 94-C Lmelt) for 2 minutes, 55 C (anneal) for 2 minutes, 72 C (extend~ for 2 minutes, this cycle was repeated 1~3 times. A final extension step was run for 10 minutes to allow for complete polymerization 3 5 of all strands. PCR product (approximately 5-10 llg~ was digested with Ndel and EcoR1 ~for CKB~ or Ndel and BamH1 ~ for CKM) and purified by electrophoresis through a 0.7% low melting point agarose TAE buffered gel (FMC BioProducts, Inc. Rockland, ME~ for cloning into expression vectors.
40 Electrophoresis was performed as described in Molecular Cloning (Sambrook, Fritsch and M~n;~ti c, 198g. Cold Spring Harbor Press, Cold Spring Har~or, NY) . Restriction WO 95112662 217 rj S ~l ~ PC~IUS94/12624 endonucleases were purchased from New England BioLabs (Beverly, MA) and digestion reactions set up as suggested by the manufacturer. DNA fragments were purified from gel slices using the f.~nf~ An kit (BiolO1, La Jolla, CA~.
s Conqtruct;n~ thf~ S;~n - ~rnreccinn Vector r-R7~8 The expression vector was constructed at Genzyme as a derivative of the plasmid pBluescript SK +/-~available from Stratagene, La Jolla, CA). Expression is 10 driven off the Lac promoter. Our vector, pRZ38, was constructed by adding an restriction enzyme site at the ATG
start codon of the b-galactosidase gene through site-directed mutagenesis. Mutagenesis protocols were followed as described using the Muta-Gene In Vitro 15 Mutagenesis Rit from BioRad (Ri rl 1 , CA) . This change in the vector allows cloning of foreign genes after the LacZ
promoter but maintains similar spacing from the promoter as in the native gene.
20 ('lnn;n~t CKM ein~ CKR nN!~ ;nt~ R7~8 Vector DNA (5 llg) was digested with Ndel and EcoR1 (for CKB) or Ndel and BamH1 (for CKM~ and gel purified as above. Approximately 100 ng digested vector DNA and 100 ng digested PCR product were ligated in a 20 111 reaction (T4 25 DNA ligase purchased from NEB, Beverly, MA) . Ligation reactions set up as described in Molecular Cloning. After overnight ;n llh~t;nn at 15 C ligation mixes were diluted to 60~1 with dlI20. 1111 of each was electroporated into electroporation competent E. coli strain MC1061. The 3 0 CeLl-Porator and Voltage Booster were purchased from Bethesda Research Labs (Bethesda, MD~ . Protocols or competent cell preparation and ele.LL~.~ v, ltion are described in the I~struction manual. Transformants were selected on-LB agar plates supplemented with 50 ,~Lg~ml ampicillin.
3 5 Transformants were analyzed for harboring the correct recombinant plasmid using the alkaline lysis miniprep t~ hn;qr1c. (Molecular Cloning) . Miniprep DNA was analyzed by restriction enzyme mapping. 25 ml cultures of pRZ52 (CKB) and pRZ53 (C~¢) were grown in LB with 5011g/ml ampicillin and 40 larger scale DNA preps were purified using QIAGEN plasmid purification columns (QIAGEN Corp., Chatsworth, CA) .
Dideoxynucleoside chain-tF~rm;nAt;nn DNA secluencing reactions WO 95112662 ~ PC~IUS94112624 2~7~a were carried out according to the standard protocols described in the Sequenase 2 O kit from USB tCleveland, OH).
[-35S]-dATP (~ew ~ngland Nuclear~ was used to radiolabel the sequences for visualization on Kodak XAR film. Reactions 5 were separated through a 6% polyacrylamide gel, the gel dried, and expbsed to ilm as described in Molecular Cloning. The DNA sequence representing the coding regions of CKs and CKM are in ~igure G1 and 2.
Once the CKM DNA sequence was ~lPtPrmin~9 to be 10 correct the a~npicillin resistance gene in pRZ53 was exchanged for the kanamycin resistance gene from Tn903 ~Nomura et al., ~ene, 3:39-51, 1987). The ampicillin gene was cut out using the restriction enzymes Ssp~ and spml, and the vector ends were blunt ended using T4 polymerase. The 15 kanamycin resistance gene had been cloned into the polylinker in a pBR322 :vector. The gene was cut out of the vector using BamH1 and was blunt ended with T4 polymerase.
The resulting C~M plasmid is referred to as pRZ69.
20 t`orc:truct; n~ the CRMl~ Co-P~ression Strain pRZ52 and pRZ69 DNA was mixed together in a concentration o~ approximately 50 llg/ml and 1 1ll of the mixture electroporated into 20 1ll MClû61 cells. The cells were plated onto 1~3 plates containing 50 llg/ml ampicillin 25 and 50 llg/ml kanamycin to select or cells that had been co-tra~sformed with both plasmids. Co-transformants were analyzed ~y restriction digestion of miniprep DNA (the standard tP~ n; q~P is described in Molecular Cloning) .
WO 95112662 ~ 1 7 5 5 9 0 pC~ S94/12624 p\nA 1 vr i n F~-~nressinn Expression analysis of the co-transformants was carried out as follows. Clones were grown overnight in 2 ml LB, 0.2~s glucose, 50 llg/ml kanamycin and 50 llg/ml S i ~71l;n at 37 C shaking. In the morning 25 ml of LB with 50 ~Lg/ml kanamycin and 50 llg/ml ampicillin was inoculated with 0 . 6 ml of the overnight culture and grown at 30 C
shaking. Cultures were grown to A600 about 0.4 then sampled at one hour intervals for 4 hours. Cultures were left 10 growing overnight and an additional sample taken. Whole cell samples were boiled in SDS-PAGE sample buffer and run through 12% polyacrylamide gels to assess protein production Gels were obtained f rom BioRad, and protocols provided with gels were followed.
Several assays were used to ~F.t~rmin~ the level and quality of the expressed r~ mhinAnt proteir,. Cultures were grown as previously described. At A600 approximately 1. 0 or af ter overnight incubation cells were harvested and resuspended in a lysis buffer (20 mM Bis-Tris pH 6.9, 0.259 20 Tween 20, 10 mM b ~ptoethanol, 10 mM EDTA, 10 mM EGTA, lmM PNSF). The resuspended cells were lysed by sonication on ice (or for larger scale analysis cells were lysed using the Microfl~ ;7~r from Microfluidics Corp., Newton, MA ) and the cell debris removed by centrifugation. Samples were 2 5 analyzed using the following assay systems after diluting into lysis buffer. The Creatine Kinase Reagent (Sigma 47-UV) is a spectrophotometric assay for kinetic determination of enzyme activity. The Creatine Phosphokinase (CPK) Isoenzymes Kit (Sigma 715-EP) separates 3 0 the various isoforms (NM, MB, and BB) and stains for activity. The CR-MB assay system for the Abbott ~c analyzer uses a microparticle enzyme i oAqsay (MEIA) to determine specific protein mass of CR-MB in a sample. (A11 protocols are provided with the assay kits.
:~uiv)- 1 ~nt ~
Those skilled in the art will recognize, or be able to ascertain, using no more than routine expGr;~ tAt;nn many equivalents to the specific embodiments 4 0 of the invention described herein. Such equivalents are ;nt.~n~ l to be ~- cced by the fallowing claims:
~O95/12662 j ' ~ PC~IUS94/12624 ~
2 ~ 8 -SEQUENCE LISTING
( l ) GENE-RAL INFORMATION:
(i~ APPLICANT: Ziegler, Robin ~.
Lon~, Sue ( ii ) TITLE OF LNVI~ I'l'LUN: Method of Producing Rl ' 'nAnt Dimeric Enzyme l O
(iii) NU~3ER OF ~U N(.~ : 4 ( iV) ~:U~ UNJ~;N~:~; ADDRESS:
(A~ ~nnR~. : Bill Gosz , Esq., 1 5 Genzyme Corporation (B) STREET: One Kendall Sc~uare ( C ~ CITY: Cambridge ( D ) STATE: MA
( E ) COUNTRY: U . S . A .
2 0 (F) ZIP: 02139 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) CC~PUTER: 1:~ PC compatible 2 5 (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l . 0, Version # l . 25 (vi) CURRENT APPLICATION DATA:
3 0 (A) APPLICATI~ NOMBER: US
( ~3 ) F ILING DATE:
( C ) CLAS S IF I CAT ION:
(viii) ATTORNEY/AGENT INFOR~TION:
3 5 (A) NAME: William G Go8z (B) REGISTRATION NOMBER: 27,787 (C) ~ ~/DOCKET NOMBER: GEN3-l0.0 WO 95/126G2 217 ~ ~ ~ O PC~/IIS941~2624 _ 9 _ ( ix) ~r~r ~ [ ~ ~ IN I ~ L l'ION INFORMATION
(A) ~rT T,T PT~nNF (617) 252--7583 (B) TELEFAX (617) 252-7600 ( C ) TELEX 2 012 23 GENCA~B
( 2 ) INFORMATION FOR SEQ ID NO
(i) SEQ~ENCE CHaRACTERISTICS
(A) LENGTH 11~6 base pairs (B) TYPE nucleic acid (C) S'rR~NlJ~l~N~S`i single (D) TOPOLOGY linear ( ii ) MnT F~'TTT~T. TYPE cDNA
(iii) ~YL~r~ L~lCAL NO
( iv) ANTI-SENSE YES
2 0 (v) FRAGMENT TYPE N-terminal (vi~ ORIGINAL SOURCE
(A) OR~ANISM Homo sapiens 2 5 ( xi ) SEQUENOE L~ ClL~ l lON SEQ ID NO l ATGCCCTTCT rr~ArAr.rrA CAACGCL~CTG AAr~rlrr~rr~rT L.~ W~ GGACGAGTTC 60 CCCGACCTGA r,rrrrrDrAA rAArrArATr~ GCCAAGGTGC ~rr.ArrrrrrA GCTGTACGCG 120 r-Arr-~Grr-rr~ rrAArAr~rAr r.rrrArrr~r TT,CACGCq'GG ACGACGTCAT rrAnArArrr 180 GTGGACAACC rr~r~rrArrr r.~rDrA~rA~r~ ACCGTGGGCT ~.1'1.1~:13 I~I.(j rr.Arr~Arr.Ar. 240 3 5 TCCTACGAAG TGTTCL~AGGA TCTCTTCGAC CCCATCATCG Ar~Arrrr.rA rrrr.rrir~rAr 300 - AAr.rrrArrn ATGACGACAA rArrrArrrr AArrrrr.ArA Arr~rrArr~ rrrrr.Arr.Ar 360 CTGGACCCC~ Ar~Arr~Tr~rT GAGcTcGr-GG GTGGCCACGG r~rrrrArrAT ~L~i~L . 4ao i~.~LLC: rrr~r~r.rAr. rrrrrrr~An rr,rrr.AnrrA TCGAGAAGCT rrrrr~rrrAA 480 WO 95/12662 . ~ PC'FIUS9411262-1 ~
2 17 ~ 0 r~ rrrTrr-Drrr rr.ArrTrr.rr. GGCCGATACT ACGCGCTCAA GAGCATGACG ~40 ~:Arrirrr.~r.r ArrDr~rDr~rT DTrr~ArnAr CACTTCCTCT Trr~DrAAr-rr ~ 600 5 ~l~ L~; rrTrr.r.f:rDT rrrrrrrr.Ar Tr~rrrr.Drr. rrr.rrrr.~AT rTrrrArDAT 660 r.ArDATAAr.A ~ l GTGGGTCAAC r.Arr.Arr.Arr ArrTrrr,r.~:T CATCTCCATG 720 r~r.AAr.rr~r7 rrAAr~Tr.AA GGAGGTGTTC ACCCGCTTCT r~rArrr~rrT CACCCAGATT 780 l O
GAAACTCTCT TrAAr.TrTAA GGACTATGAG TTCATGTGG,A ACCCTCDCCT GGGCTACATC 840 CTCACCTGCC CATCCAACCT rrr.rDrrr,r~ rTr,rrrr,r~r. GTGTCGATAT CAAGCTGCCC 900 nr~rDr~r~r~rG GTGTGGACAC ~l~ , TCGACGTCTC rD~rrr~TnAr 1020 ~l~iW-r TCTCAGAGGT r~nAnrTr~r~Tr~ CAGATGGTGG 'rr~GACGGAGT rADrrTrrTr 1080 ATCGAGATGG AACAGCGGCT rr.Drr~rrr.r CAGGCCATCG ACGACCTCAT r~rrTr~rrrAr~ 1140 AAATGA 119.6 ~IVO 95112662 2 1 7 5 ~ ~ ~ PC~IUS94l12624 1 ~ _ (2 ) INFORM~TION FOR SEQ ID NO: 2 ( i ) SEQ~ENCE CH~RACTERISTICS:
~A) LENGT~: 1146 base ~airs ~B) TYPE: nucleic acid ~ C ) S'T'T~ A N I I ~ 'i 'i: s irlgle ~D) ~OPOLOGY: linear ~ ii) M~T,T~`TTTT~' TYPE: c~A
l O
(iii) ~Ul'~ll~AL: NO
(iv) P~NTI-SENSE: YES
(v) FRAG~ENT TYPE: N-ter~ninal ( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ATGCCATTCG r.~AArArrrA CAACAAGTTC AAGCTGAATT ArAAnrrTnA rr.Arr.Ar.~Ar 60 CCCGACCTCA rrAA~rA~AA CAACCACATG r~rrAAr~r TGACCCTTGA ACTCTACAAG 120 AAGCTGCGGG ArAArr.~r.A~ CCCPLTCTGGC TTCACTGTA~ ACGATGTCAT rrAr.ArAr.r.A 180 2 5 GTGGACAACC CAGGTCACCC CTTCATCATG ACCGTGGGCT ~L~L~ TGATGAGGAG 240 TCCTPLCGAAG TTTTCAAGGA ACTCTTTGAC CCCATCATCT CGGATCGCCA rrrr~rr~Ar 3 o O
AAArrrAr~r. ArAArrAr~ GPCTGACCTC AArrA'rGAAA ACCTCAAGGG TGGAGACGAC 360 CTGGACCCCA Ar~rArr.~rr~ rAr.rArrrrr, GTCCGCPLCTG rrrrrArrAq~ rAAr~rr~rAr 420 ACGTTGCCCC CACACTGCTC CCrTGGCGAG ~ TGGAGAAGCT rTr~r.~r~AA 480 3 5 r.r~r~rrAArA GCCTGACGGG CGAGTTCAAA rrr.AAr.~Ar~ ACCCTCTGAA r.ArrA~r.Arr. 5~0 rAr.AArr.Arr AGCAGCAGCT CATCGATGAC CACTTCCAGT TCGACA~GCC WL~ U~ 600 ,~-~ CCTCAGGCAT rr,rrrrrrAr llr~r~rrrr~rr~ CCCCTGGCAT CTGGCACAAT 660 r.ArAAr~Ar.A ~7~lL~,l~il G~:GTGAAC GAGGAGGATC ACCTCCGGGT CATCTCCATG 720 W095112662 , PC~IUS9~112624 - 217~9a ~ 12-r~r.~r~rr~ GCAACATGAA CCZ~_LLLL~ - ~.~ I 1.~ 1 I~ I GCGTAGGGCT GCAGAAGATT 780 GAG~AGATCT TTAAGAAAGC TrrrrD.rrrr TTCI!TGTGaA ArrDrrArr~r GGGCTACGTG 840 Sr~rsrr~rrr cATccAAr-cT r.rrr~r~r.rr. r~rrnTrr.~r. GCGTGCATGT GAAGCTGGCG 900 r~rrTr~r~r~ ~r~rrrr~ GTTCGAGGAG ATCCTCACCC 4~Ll~i~4L~ rr~r~r~r. 960 rrl~r~rr.~r. CGGTGGACAC AGCTGCCGTG rrr~rrl~r.~rAT TTGACGTGTC CAACGCTGAT 1~20 CGTCCGAAGT Ar.~r~rrl~r. CAGCTGGTGG TGaATGGTGT GAAGCTCATG 1080 GTGC~AA.;~TGG AGAAGAAGTT rr.~r~ rr~r CAGTCCATCG ~rr.P~r~r~ rrrrnrrr~r. , 140 WO 95/12662 2 t 7 5 5 ~ O PC~IUS9*12624 ( 2 ) INFOKM~TION FOR SEQ ID NO: 3:
( i ) sEQr~ENcE rr~R ~
(A) LEWGTH: 381 amino acids (B) TYPE: arnino acid (C ) s~R~Nnr~r~NF ~.~: single (D) TOPOLOGY: linear (ii) Mr,r,r~(~rrr,r~ TYPE: protei~L
(v) FRAGMENT TYPE: N-terminal (xi) sEslrJENcE L~ KLL~L~ L~I: SEQ ID NO:3:
1 5 Iet Pro Phe Ser ~sn Ser His Asn Ala Leu Lys Leu Arg Phe Pro Al~
Glu Asp Glu Phe Pro A~P Leu Ser Ala His Asn Asn His ~et Ala Lys Val Leu Thr Pro Glu Leu Tyr Ala Asp Val Arg Ala Ly~ Ser Thr Pro Ser Gly Phe Thr Leu Asp Asp Val Ile Gln Thr Gly Val Asp Asn Pro 2 5 so ss 60 Gly His Pro Tyr Ile Yo~ Thr V~l Gly Cys V.ll Ala Gly Asp Glu Glu 6s 70 75 80 3 0 Ser Tyr Glu V~l Phe Lys Asp Leu Phe Asp Pro Ile Ile Glu Asp Arg His Arg Arg Tyr Lys Pro Ser AsP ASp Asp Lys Thr AsP Leu Asn Pro Asp Asn Leu Gln Gly Gly A~p Asp . Leu Asp Pro Asn Tyr Val Leu Ser 4 0 Ser Arg Val Ala Thr Gly Arg Ser Ile Arg Gly Phe Cys Leu Pro Pro WO 95/12C62 . ' ,` ' ` PCI!/US94112624~
- 21~9~ 14_ His Cys Ser Arg Gly Glu Arg Arq Ala Ile Glu Lys Leu Alo Val Glu l~S lS0 lSS 160 Ala Leu Ser Ser Leu Asp Gly Asp Leu Ala Gly Arg Tyr Tyr Ala Leu Lys Ser Met Thr Glu A1~ Glu Gln Gln Gln Leu Ile Asp Asp His Phe 0 Leu Phe Asp LYB Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala l9S 200 205 Arg Asp Trp Pro Asp Al~ A121 Ars~ Ile Trp His Asn Asp Asn Lys Thr Phe Leu Val Trp Val Asn Glu Glu Asp E~is Leu Arg Val Ile Ser Met Gln Lys Gly Gly Asn Met Lys Glu Val Phe Thr Arg Phe Cys Thr Gly Leu Thr Gln Ile Glu Thr Leu Phe Lys Ser Lys Asp Tyr Glu Phe Met 2 5 Trp Asn Pro His Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn Leu Gly Thr Gly Leu Arg Ala Gly Val Asp Ile Lys Leu l?ro Asn Leu Gly Lys His Glu Lys Phe Ser Glu Val Leu Lys Arg Leu Arg heu Gln Lys Arg Gly Thr Gly Gly Val Asp Thr Ala Ala Val Gly Gly Val Phe Asp Val Ser Asn Ala Asp Arg Leu Gly Phe Ser Glu Val Glu Leu Val Gln Met 4 0 Val V~l Asp Gly Val Lys Leu Leu Ile Glu Met Glu Gln Arg Leu Glu ~ WO 95112662 ~17 ~ 5 9 0 rc~Nsg4/12624 ~ 15 --Gln Gly Gln Ala Ile Asp Asp ~eu D~et Pro Al~ Gln Lys l O
~0 ~Vo 95/12662 , ~ ' , PCFIUS94112624 ~
21~S5~ - t6-( 2 ) rNFORMATION FOR SEQ m NO: 4:
( i ) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 381 a~ino acids /`
(B) TYPE: amino acid (C) Slr1?1~N~ N~:~;';: single (D) TOPOLO(~Y: linear (ii) MOT T~'TIT,T. TYPE: protein l O
(v) FR~T TYPE: N-terminal (xi ) SEQUENCE L~ ~L~L~16~N: SEQ ID NO: 4:
Met Pro Phe Gly Asn Thr His Asn Lys Phe Lys Leu Asn Tyr Lys Pro Glu Glu Glu Tyr Pro Asp Leu Ser Lys His Asn Asn His Met Al~ Lys Vel Leu Thr Leu Glu Leu Tyr Lys Lys Leu Arg Asp Lys Glu Ile Pro Ser Gly Phe Thr Vzll Asp Asp VP1 Ile Gln Thr Gly V~l Asp Asn Pro 2 5 so s5 60 Gly His Pro Phe Ile Met Thr Val Gly Cys Val Al~ Gly Asp Glu Glu 6s 70 75 80 3 0 ger Tyr Glu VP1 Phe Lys Glu Leu Phe Asp Pro Il~ l:le Ser Asp Arg His Gly Gly Tyr Lys Pro Thr Asp Lys His Lys Thr Asp Leu Asn His 100 105 ~ 110 Glu Asn Leu Lys Gly Gly Asp Asp Leu Asp Pro Asn Tyr V~l Leu Ser Ser Pro V- 1 Ar~ Thr Gly Arg Ser Ile ~ys Gly Tyr Thr Leu Pro Pro WO95112662 2~7~59D PC~IUS94112624 His Cys Ser Arg Gly Glu Arg Arg Ala Val Glu Lys Leu Ser Val Glu Ala Leu Asn Ser ~eu Thr Gly Glu Phe Lys Gly Lys Tyr Tyr Pro Leu Lys Ser Met Thr Glu Lys Glu Gln Gln Gln Leu Ile Asp Asp His Phe 0 Gln Phe Asp Lys Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala Arg His Trp Pro Asp Al~ Pro Gly Ile Trp His Asn Asp Asn Lys Ser l S
Phe Leu Val Trp Val Asn Glu Glu Asp His Leu Arg Val Ile Ser Met Glu Lys Gly Gly Asn Net Lys Glu Val Phe Arg Arg Phe Cys Val Gly 2 0 2~5 250 255 Leu Gln Lys Ile Glu Glu Ile Ph~ Lys Lys Ala Gly Hia Pro Phe Met 260 265 270 ~~
2 5 Trp Asn Gln His Leu Gly Tyr Val Leu Thr Cys Pro Ser Asn Leu Gly Thr Gly Leu Arg Gly Gly Val His Val Lys Leu Ala His Leu Ser Lys His Pro Ly~i Phe Glu Glu Ile Leu Thr Arg Leu Arçr Leu Gln Lys Arg Gly Thr Gly Ala Val Asp Thr Ala Ala Val Gly Ser Val Phe Asp Val er ~sn Ala Asp Arg Leu Gly Ser Ser Glu Val Glu Gln Val Gln Leu 4 0 Val Val Asp Gly Val Lys Leu Met Val Glu Met Glu Lys LYs Leu Glu WO 95/12662 , ~ PC~IUS94/;2624 21~5~a - 18 -Lys Gly Gln Ser Ile Asp Asp Met 'fle Pro ~1~ Gln Ly~
Claims (16)
1. A method of producing an eukaryotic heterodimeric enzyme in an active form, comprising:
(a) constructing:
(i) a first DNA vector containing DNA encoding a first subunit of the enzyme; and (ii) a second DNA vector containing DNA encoding a second subunit of the enzyme;
(b) transforming a prokaryotic host with:
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed prokaryotic host under conditions appropriate for the expression of the dimeric enzyme.
(a) constructing:
(i) a first DNA vector containing DNA encoding a first subunit of the enzyme; and (ii) a second DNA vector containing DNA encoding a second subunit of the enzyme;
(b) transforming a prokaryotic host with:
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed prokaryotic host under conditions appropriate for the expression of the dimeric enzyme.
2. The method of Claim 1 wherein the eukaryotic heterodimeric enzyme is mammalian.
3. The method of Claim 1 wherein the eukaryotic heterodimeric enzyme is a kinase.
4. The method of Claim 3 wherein the kinase is a creatine kinase.
5. The method of Claim 1 wherein:
(a) the first subunit is creatine kinase subunit B and second subunit is creatine kinase subunit M; or (b) the first subunit is creatine kinase subunit M and second subunit is creatine kinase subunits.
(a) the first subunit is creatine kinase subunit B and second subunit is creatine kinase subunit M; or (b) the first subunit is creatine kinase subunit M and second subunit is creatine kinase subunits.
6. The method of Claim 1 wherein the prokaryotic host is bacterial.
7. The method of Claim 6 wherein the bacterial host is a Escherichia.
8. A method of producing a human dimeric enzyme comprising:
(a) constructing:
(i) a first DNA vector containing DNA encoding a first subunit of the enzyme; and (ii) second DNA vector containing DNA encoding a second subunit of the enzyme;
(b) transforming a prokaryotic host with:
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed prokaryotic host under conditions appropriate for the expression of the heterodimeric enzyme.
(a) constructing:
(i) a first DNA vector containing DNA encoding a first subunit of the enzyme; and (ii) second DNA vector containing DNA encoding a second subunit of the enzyme;
(b) transforming a prokaryotic host with:
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed prokaryotic host under conditions appropriate for the expression of the heterodimeric enzyme.
9. The method of Claim 8 wherein the human dimeric enzyme is a kinase.
10. The method of Claim 9 wherein the kinase is creatine kinase.
11. The method of Claim 8 wherein the prokaryotic host is bacterial.
12. The method of Claim 11 wherein the bacterial host is a Escherichia.
13. The method of Claim 8 wherein the first and second subunit are creatine kinase subunit B.
14. The method of Claim 8 wherein the first and second subunit are creatine kinase subunit M.
15. A method of producing a creatine kinase enzyme comprising:
(a) constructing:
(i) a first DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO.: 1; and (ii) a second DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO. :2;
(b) transforming a bacterial host with:
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed bacterial host under conditions appropriate for the expression of the dimeric enzyme.
16. A method of producing a creatine kinase enzyme comprising:
(a) constructing:
(i) a first DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO. :2; and (ii) a second DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO. :1;
(b) transforming a bacterial host with.
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed bacterial host under conditions appropriate for the expression of the dimeric enzyme.
17. An eukaryotic heterodimeric enzyme produced by the method of Claim 1.
18. An human dimeric enzyme produced by the method of Claim 8.
19. A creatine kinase enzyme produced by the method of Claim 15.
20. A creatine kinase enzyme produced by the method of Claim 16.
21. A transformed prokaryotic host produced by the method of Claim 1.
22. A transformed prokaryotic host produced by the method of Claim 8.
23. A transformed prokaryotic host produced by the method of
15. A method of producing a creatine kinase enzyme comprising:
(a) constructing:
(i) a first DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO.: 1; and (ii) a second DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO. :2;
(b) transforming a bacterial host with:
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed bacterial host under conditions appropriate for the expression of the dimeric enzyme.
16. A method of producing a creatine kinase enzyme comprising:
(a) constructing:
(i) a first DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO. :2; and (ii) a second DNA vector containing DNA encoding a DNA sequence selected from all or a portion of the DNA sequence of SEQ ID NO. :1;
(b) transforming a bacterial host with.
(i) the first DNA vector; and (ii) the second DNA vector; and (c) culturing the transformed bacterial host under conditions appropriate for the expression of the dimeric enzyme.
17. An eukaryotic heterodimeric enzyme produced by the method of Claim 1.
18. An human dimeric enzyme produced by the method of Claim 8.
19. A creatine kinase enzyme produced by the method of Claim 15.
20. A creatine kinase enzyme produced by the method of Claim 16.
21. A transformed prokaryotic host produced by the method of Claim 1.
22. A transformed prokaryotic host produced by the method of Claim 8.
23. A transformed prokaryotic host produced by the method of
Claim 15.
24. A transformed prokaryotic host produced by the method of
24. A transformed prokaryotic host produced by the method of
Claim 16.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14665593A | 1993-11-02 | 1993-11-02 | |
| US08/146,655 | 1993-11-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2175590A1 true CA2175590A1 (en) | 1995-05-11 |
Family
ID=22518370
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2175590 Abandoned CA2175590A1 (en) | 1993-11-02 | 1994-11-02 | Method of producing recombinant dimeric enzyme |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0729505A1 (en) |
| JP (1) | JPH09504698A (en) |
| AU (1) | AU8131994A (en) |
| CA (1) | CA2175590A1 (en) |
| WO (1) | WO1995012662A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6174715B1 (en) * | 1996-06-14 | 2001-01-16 | Toyota Jidosha Kabushiki Kaisha | Prenyl diphosphate synthetase genes |
| US5981249A (en) * | 1998-02-05 | 1999-11-09 | Spectral Diagnostics, Inc. | Single-chain polypeptides comprising creatine kinase M and creatine kinase B |
| JP2001299361A (en) * | 2000-04-28 | 2001-10-30 | Oriental Yeast Co Ltd | Recombinant human creatine kinase heterodimer with solution stability |
| GB0203552D0 (en) * | 2002-02-14 | 2002-04-03 | Beaumont Nicholas J | Peptide growth factor |
| DE102004013843A1 (en) * | 2004-03-20 | 2005-10-13 | Degussa Ag | Expression of nitrile hydratases in the two-vector expression system |
-
1994
- 1994-11-02 AU AU81319/94A patent/AU8131994A/en not_active Abandoned
- 1994-11-02 WO PCT/US1994/012624 patent/WO1995012662A1/en not_active Application Discontinuation
- 1994-11-02 CA CA 2175590 patent/CA2175590A1/en not_active Abandoned
- 1994-11-02 EP EP95900517A patent/EP0729505A1/en not_active Withdrawn
- 1994-11-02 JP JP7513402A patent/JPH09504698A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0729505A1 (en) | 1996-09-04 |
| AU8131994A (en) | 1995-05-23 |
| WO1995012662A1 (en) | 1995-05-11 |
| JPH09504698A (en) | 1997-05-13 |
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