United States Patent [191 [11] Patent Number: 4,831,120 Aviv et al. [45] Date of Patent: May 16, 1989

[54] METHOD FOR RECOVERING A PURIFIED ANIMAL GROWTH HORMONE OR FOREIGN PATENT DOCUMENTS POLYPEPTIDE ANALOG THEREOF FROM 0061250 9/1982 European Pat. Off. . A BACTERIAL CELL 0127305 12/1984 European Pat. Off. . 2083824 3/1982 United Kingdom . [75] Inventors: Haim Aviv; Marian Gorecki, both of 8304418 12/ 1983 United Kingdom . Rehovot; Avigdor Levanon, Netania; 2138004 9/1984 United Kingdom . Amos Oppenheim, Jerusalem; Tikva Vogel, Rehovot; Elisha Zeelon, OTHER PUBLICATIONS Hashiva; Menachem Zeevi, Ramat Principles of Biochemistry, Lehninger 1982, p. 177. Gan, all of Primary Examiner—Johnnie R. Brown [73] Assignee: Bio-Technology General Corp., New Assistant Examiner—Garnette D. Draper York, NY. Attorney, Agent, or Firm-John P. White [21] Appl. No.: 752,441 [57] ABSTRACT An improved vector upon introduction into a suitable [22] Filed: Jul. 3, 1985 bacterial host containing the thermolabile repressor C1 renders the host cell capable, upon increasing the tem Related U.S. Application Data perature of the host cell to a temperature at which the repressor is destroyed, of effecting expression of a de [63] Continuation of Ser. No. 514,188, Jul. 15, 1983, aban doned. sired gene inserted into the vector and production of polypeptide encoded by the gene. The vector is a dou [30] Foreign Application Priority Data ble-stranded DNA molecule which includes in 5' to 3’ order the following: a DNA sequence which contains Jul. 3, 1984 [EP] European Pat. Off...... 84107717.5 the promoter and operator PLOL from lambda bacterio [51] Int. Cl.4 ...... _ ...... C07K 3/28 phage; the N utilization site for binding antiterminator [52] U.S. Cl...... 530/399; 530/412; N produced by the host cell; a DNA sequence 530/416; 530/417; 530/418; 530/422; 530/820; which contains a ribosomal binding site for rendering 530/825; 530/427; 435/68 the mRNA of the desired gene capable of binding to [58] Field of Search ...... 530/399, 412, 416, 4l7—418, ribosomes within the host cell; an ATG initiation codon 530/422, 427; 435/68 or a DNA sequence which is converted into an ATG initiation codon upon insertion of the desired gene into [56] References Cited the vector; a restriction enzyme site for inserting the U.S. PATENT DOCUMENTS desired gene into the vector in phase with the ATG initiation codon; and additionally a DNA sequence 4,426,323 1/1984 Jain ...... 435/68 which contains an origin of replication from a bacterial 4,512,922 4/1985 Jones et al. 435/68 plasmid capable of autonomous replication in the host 4,612,367 9/1986 Grinnan et a1. 530/413 cell and a DNA sequence which contains a gene associ 4,656,255 4/1987 Seely ...... , ...... 530/417 4,658,021 4/ 1987 Goeddal et al. . 530/ 399 ated with a selectable or identifiable trait which is mani 4,677,196 6/1987 Rausch et a1. .. . 530/416 fested when the vector is present in the host cell.

4,693,973 9/1987 Buell ...... 530/399 4,728,609 8/1988 Bhatt et a1...... 435/68 11 Claims, 15 Drawing Sheets

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4,831,120 1 2 Numerous reports may be found in the literature METHOD FOR RECOVERING A PURIFIED concerning the cloning of eucaryotic genes in plasmids ANIMAL GROWTH HORMONE OR containing the PL promoter from )t bacteriophage. (Ber POLYPEPTIDE ANALOG THEREOF FROM A nard, H. V. et al., Gene (1979) 5, 59; Derom, C. et al., BACTERIAL CELL Gene (1982) 17, 45; Gheysen, D. et al., Gene (1982) 17, 55; Hedgpeth, J. et al., Mol. Gen. Genet. (1978) 163, This is a continuation of application Ser. No. 514,188, 197; Remaut, E. et al., (1981) Gene 15, 81; and Derynck, ?led July 15, 1983 now abandoned. R., et al., Nature (1980) 287, 193. In addition, European Patent Application No. 041.767, published Dec. 16, BACKGROUND OF THE INVENTION 1981 describes expression vectors containing the PL One aspect of involves the inser promoter from 1» bacteriophage. However, none of tion of foreign DNA sequences derived from eukary these references describe the use of the C11 ribosomal otic sources into or other microorgan binding site. isms. A further re?nement of genetic engineering con The use of a vector containing the PL promoter from cerns inducing the resulting microorganism to produce 15 A bacteriophage and the C11 ribosomal binding site has polypeptides encoded by the foreign DNA. Production been described. (Oppenheim, A. B. et al., J. Mol. Biol. of polypeptides can be considered a two-step process, (1982) 158, 327 and Shimatake, H. and Rosenberg, M., with each step including numerous substeps. The two Nature (1981) 292, 128.) These publications describe the steps are transcription and . To produce a production of increased levels of C11 protein but do not polypeptide efficiently and in quantity both steps of the 20 involve or describe the production of eucaryotic prote process must be efficient. Transcription is the produc ins. tion of mRNA from the gene (DNA). Translation is the In 1982 Shatzman and Rosenberg presented a poster production of polypeptide from the mRNA. at the 14th Miami Winter Symposium (Shatzman, A. R. A critical substep of the transcription process is initia and Rosenberg, M., 14 Miami Winter Symposium, ab tion, that is, the binding of RNA polymerase to a pro 25 stract p98 [1982]). This abstract provides a non~enabling moter-operator region. The sequence of deoxyribonu disclosure of the use of a vector containing PL from A cleotide bases which make up the promoter region may bacteriophage, Nut and the C11 ribosomal binding site to vary and thereby effect the relative ef?ciency of the synthesize a “eucaryotic” polypeptide (SV40 small T promoter. The ef?ciency depends on the affinity of the antigen is actually not a eucaryotic polypeptide but a RNA polymerase for the promoter. 30 viral protein) in an amount greater than 5% of the cell The ef?ciency of translation is affected by the stabil protein in an unnamed bacterial host. The operator used ity of the mRNA. Increased stability of the mRNA is not de?ned. Neither an origin of replication nor a permits improved translation. Although the exact deter gene for a selectable phenotype is identi?ed. This sys minants of mRNA stability are not precisely known, it is tem with which the vector is used is described as includ known that mRNA secondary structure as determined 35 ing certain host lysogens into which the vector can be by the sequence of its bases has a role in stability. stably transformed. The present invention in one em The initial substep of translation involves binding of bodiment, i.e., pMGlOO, may have certain similarities to the ribosome to a base sequence on the mRNA known this vector. However, it is not transformed into a host as the Shine-Dalgarno sequence or the ribosomal bind lysogen, but rather into suitable E. coli host strains ing site (RBS). The synthesis of polypeptides begins which contain the thermolabile repressor C1 and the N when the ribosome migrates along the mRNA to the gene but from which the rest of the lysogen has been AUG start codon for translation. Generally these co removed. Moreover, it has been employed to produce dons are found approximately 10 bases “downstream” bGH and hGI-I analogs in amounts in excess of 20% of from the Shine-Dalgarno site. Factors which increase total cell protein. the ef?ciency of translation include those which en 45 In addition, in other embodiments of this invention hance binding of the ribosomes to the Shine-Dalgarno ribosomal binding sites which differ from C11 are em site. It has been shown that the secondary structure of ployed. Also, in the presently most preferred vectors, the mRNA in the region of the Shine-Dalgarno se pNDS and its derivatives, nonessential sequences have quence and the AUG codon and the distance between been removed to create a vector permitting polypeptide the Shine-Dalgarno sequence and the AUG codon each 50 production in amounts which are more than 10% play a critical role in determining the ef?ciency of trans greater than those obtained with pMGlOO. lation. Other factors which affect the ef?ciency of Recently, applicants have learned of the existence of translation are premature termination and attenuation. a pending US. patent application in the name of M. Efficiency of translation can be improved by removing Rosenberg ?led under Ser. No. 457,352 now US. Pat. the attenuation sites. 55 No. 4,578,355 by the National Institutes of Health, A difficulty encountered in attempts to produce high Dept. of Health and Human Services; USA. Portions amounts of eukaryotic polypeptides in bacterial cells of this application have been obtained from the National involves the inability of cells producing large amounts Technical Information Service, US. Dept. of Com of mRNA to grow ef?ciently. This dif?culty can be merce. However, the claims are not available and are eliminated by preventing transcription by a process maintained in con?dence. The available portions of the known as repression. In repression genes are switched application have been reviewed. This disclosure is not off due to the action of a protein inhibitor (repressor enabling. It indicates that the host is important (p8, line protein) which prevents transcription by binding to the 17) but fails to identify any suitable host. It further operator region. After microorganisms have grown to depends upon the use of a A mutant which is not speci desired cell densities, the repressed genes are activated 65 ?ed (p4, line 20). It indicates that the host contains by destruction of the repressor or by addition of mole lysogens (p8, line 18) unlike the present invention in cule known as inducers which overcome the effect of which the host is not lysogenic. It mentions cloning and the repressor. expression of a eucaryotic gene, monkey metalloth 4,831,120 3 4 ionein gene, (p7, line 18) but does not provide details. It polypeptide produced. Preferred plasmids for bGH are speci?es that neither the sequence nor the position of pRec 5 and pROl 1; and for hGH, pTV 18(1) and pTV any nucleotide in the C11 ribosomal inding region has 104(2). Suitable hosts include Escherichia coli A1637, been altered. (p3, line 27) In the present invention such A1645, A2602 and A1563; A1637 being presently pre alteration is possible. ferred. No disclosure is present in the art concerning: suc The resulting host vector systems can be employed to cessful expression with a PL-CU containing vector sys manufacture polypeptides. The host cells containing the tem of bovine or human growth hormones; production plasmids are grown under suitable conditions permit of bGH or hGH analogs having biological activity; ting production of polypeptide and the resulting poly compositions containing such analogs or uses of them; peptide is recovered. Presently preferred conditions or induction methods for achieving polypeptide pro involve growth at about 42° C. for 10 to 30 minutes, duction in amounts greater than 20% of the total pro particularly 15 minutes, followed by continued growth tein produced by the host. at about 37°-39° C. for sufficient time to make the total The only disclosure in the art concerning production growth period about 60-90 minutes, particularly of bGH analogs by hosts transformed with genetically 5 growth at 38°-39° C. for about 75 minutes. Presently engineered vectors involves the use of the Trp pro preferred growth media are lactalbumin hydrolysate moter to produce a bGH analog having the amino acid with addition of glucose or brain heart infusion. Met at the N-terminus of the phenylalanine form of Using the host-vector systems, analogs of bGH and natural bGH (Seeburg, P. H. et al., DNA (1983) 2, 37. hGH have been prepared. These analogs may be incor The only disclosure in the art concerning production porated into veterinary or pharmaceutical composi of hGH analogs by hosts transformed with genetically tions, respectively. The respective analogs directly, or engineered vectors involves the use of the Lac and Trps in such compositions, may be used to stimulate milk or promoters to produce an analog of hGI-I having the meat production in a bovine or to treat human growth amino acid Met at the N-terminus of the natural hGH. hormone de?ciency. (Goedell, D. V. et al., Nature (1979) 281, 544). 25 DESCRIPTION OF THE FIGURES FIG. 1. Construction of pMGlOO expression vector. SUMMARY OF THE INVENTION This plasmid was built by inserting a fragment of )\ This invention concerns an improved expression vec phage DNA contained between restriction sites HaeIII tor which upon introduction into a suitable bacterial (location 38150) and Sau3a (location 38362) into a host cell, namely, Escherichia coli, containing the ther pKC30 plasmid DNA cleaved with Hpal and BamHl. molabile repressor C1 renders the host cell capable, The HaeIlI-Sau3a fragment carries nutR, tR1, cy' and upon increasing the temperature of the host cell to a ribosomal binding site of C]; protein (CH-RES). Sub temperature at which the repressor is destroyed, of cloning of the CH-RBS containing DNA into pKC30 effecting expression of a desired gene inserted into the creates pMGlOO which contains a unique BamHl re vector and production of the polypeptide encoded by striction site right after the ATG initiation codon of the gene comprising: CH-RBS and an Ndel restriction site within the ATG a double-stranded DNA molecule which includes in 5’ triplet (bottom inset). Numbers in parentheses denote to 3' order the following: location of restriction sites on the A phage DNA. a DNA sequence which contains the promoter and FIG. 2. Construction of pRec % plasmid. A bGH operator PLOL from lambda bacteriophage; 40 cDNA containing plasmid, D4 was digested with HaeII. the N utilization site for binding antiterminator N pro A resulting 1600 bp large fragment was puri?ed and tein produced by the host cell; subjected to digestion at 37° C. for 5 minutes with 5 a DNA sequence which contains a ribosomal binding units of S1 exonuclease. A synthetic EcoRl linker with site for rendering the mRNA of the desired gene the sequence: capable of binding to ribosomes within the host cell; 45 an ATG initiation codon or a DNA sequence which is GGAATTCC converted into an ATG initiation codon upon inser CCTTAAGG tion of the desired gene into the vector; and a restriction enzyme site for inserting the desired gene into the vector in phase with the ATG initiation co was attached by ligation. The product was cleaved with don; EcoRl and inserted into pBR322 which had been and which additionally includes a DNA sequence cleaved with EcoRl. A clone, pALRl, was isolated which contains an origin of replication from a bacte which upon cleavage with EcoRl release a 1200 bp rial plasmid capable of autonomous replication in the fragment with the sequence: host cell and a DNA sequence which contains a gene associated with a selectable or identi?able phenotypic AATTCCCA . . .

trait which is manifested when the vector is present in GGGT . . . the host cell. Preferred vectors are pMG 100 and pNDS. Genes, i.e., cDNAs, encoding desired polypeptides at the 5' end. Formation of this sequence demonstrates such as growth hormones, e.g., bovine, porcine, that pALRl contains an EcoRl restriction site which chicken or human growth hormones, superoxide dismu includes the TTC codon for residue number 1 (phenyl tase, apoprotein E, viral protein 1 of foot and mouth alanine) of authentic bGH. pALRl was subjected to a disease virus, protein A from S. aureus, interleukin III, partial cleavage with Pstl. The digest was ligated with an enzyme or analogs thereof may be inserted into the HindIII linkers and cleaved with EcoRl and HindIII. restriction enzyme site of the vector to create plasmids. The fragment containing bGH cDNA was isolated and The plasmids in turn can be introduced into suitable subcloned into pBR322 between EcoRl and HindIII hosts where the genes can be expressed and the desired restriction sites to give pALSOO. The subcloned bGH 4,831,120 5 6 cDNA fragment was then excised from pAL500 with hGH is an analog missing the ?rst 13 amino acid resi EcoRl and HindIII, “?lled in” with DNA polymerase dues and having at its Nterminus Met14. “Klenow” fragment and inserted into the pMGlOO ex Plasmid pTV 18(1) was partially digested with Ndel pression vector (FIG. 1) opened at the BamHI site and and ligated with a synthetic linker which contains the also “?lled in” as above. The resulting vector pREC 5 codons for amino acids 1-13 of hGH:

TATGTTCCCAACCATTCCATTATCCCGTCTGTTCGACAACGC ACAAGGGTTGGTAAGGTAATAGGGCAGACAAGCTGTTGCGAT. 2/2, expresses a modi?ed bGH which is altered at its amino terminus as follows: The linker is also complementary to the Ndel site on pTV 18(1) and positions the complete hGH gene in MetAspGlnPhelProz. . . bGH phase with the ATG initiation codon of the pND5 ex pression vector (FIG. 3). Thus, the resulting plasmid, The plasmid pREC 2/2 was digested with Pstl and the pTV 104(2), expresses native hGH with an extra methi fragment containing the PL promoter and the 5’ end of onine at the N-terminus. the bGH gene (designated fragment A) was isolated. FIG. 5 shows the vector pAL Trp 46 which contains This fragment was ligated to a Pstl fragment from pAL the Trp promoter and the ?rst seven amino acids of the 500 (designated fragment B). The then resulting vector, Trp E gene transcriptionally fused to the B-galactosi pRec 5, expresses a modi?ed bGH which is altered at its 20 dase gene. amino terminus as follows: FIGS. 6, 7 and 8 show a series of expression vectors (Tac) containing a part of the Trp promoter and Lac MetAspGlnPhelli’ro2 . . . bGH operator followed by restriction sites for insertion of a FIG. 3. Construction of expression vectors pND5, desired gene and expression of bGH under the control 25 of Tac promoter. pND55 and pROll. A plasmid pOG7 (A. Oppenheim, FIGS. 9 and 10 show expression vectors containing S. Gottesman and M. Gottesman, J. Mol. Biol. (1982) 158, 327) was cleaved with Ndel. The ends of the large bGH cDNA under the control of the histidine pro fragment carrying the PL promoter nutL, t}; and C11 moter. RBS were ligated to give the pND5 expression vector. FIG. 11 shows insertion of the bGH gene into an This pND5 vector DNA is opened with Ndel. Insertion 30 expression vecto under the control of the Lac promoter. FIG. 12 shows expression of bGH gene under control of that Ndel fragment from pRec § (FIG. 2) which of Omp F promoter. contains bGH cDNA results in a plasmid pROll which FIG. 13. Construction of Met4-bGH analog pAL401 appears to be a better expressor of the modi?ed bGH described in FIG. 2 than pRec §. Insertion of synthetic and expression vectors pND6 and pNDll with altered restriction sites: linkers with the sequence: 35 pAL401 which expresses a modi?ed form of bGH which is lacking the ?rst three amino acids at the amino TATGAGCTCA terminus of the bGH (Met4 bGH) was constructed by ACTCGAGTAT triple ligation of the following: 40 (a) a bGH DNA fragment of 623 base pairs with PvuII into pOG7 cleaved with Ndel results in an expression and HindII excised from pALSOO vector pND55 which contains a unique Sacl restriction (b) a linker formed by synthesizing two DNA strands whgich after puri?cation were annealed to form:

CCATATGTCCTTGTCCGGCCTGI I IGCCAACGCTGTGCT GCGACACGAGGCCCGAGTCGTGGACGTGGTCGACG site in front of ATG. When pND55 is cleaved with Sacl and treated with DNA polymerase “Klenow” fragment an ATG initiation codon results which follows the PL promoter and CH-RBS. This vector is suitable for ex 50 which was “?lled in” with DNA polymerase “Klenow” pression of a wide variety of eukaryotic genes lacking fragment and then cleaved with Ndel and PvuII to an ATG initiation codon. prepare a 58 base pair fragment which was recovered FIG. 4 Construction of pTV 18(1) and pTV 104(2). A and puri?ed. plasmid, pTVHGI-I was prepared by cloning cDNA (c) pNDll which was prepared as follows. An expres encoding hGH into the HindIII site of pBR 322 using 55 sion vector pOG7 was altered by elimination of Hin standard methods. Meth. Enzymol. (1979) 68, 75. This dIII and one of the Ndel sites (distant from the ATG plasmid was digested with HindIII. The resulting 800 initiator codon) to obtain pND6. Then HindIII link base pair fragment was puri?ed and further digested ers were introduced into a Sall site to give pNDll. with FnuDII and “?lled in” with DNA polymerase FIG. 14. Construction of authentic bGH modi?ed “Klenow” fragment. This treatment removes codons with methionine at the amino terminus and various for the ?rst 16 amino acids of hGH. The resulting DNA analogs of bGH. fragment is ligated with a synthetic linker which re (a) Plasmid pAL40l is treated with Ndel. A synthetic stores the codons for the sequence of hGH from Met14 DNA linker containing an ATG initiation signal and and regenerate an Ndel restriction site in front of the the code for the ?rst three amino acids at the amino ATG codon for Metl‘l. After treatment with Ndel this 65 terminus of native bGH is ligated into the Ndel site. semisynthetic DNA was inserted into the pND5 vector The resulting vector pAL60l leads to the expression opened with Ndel. The resulting plasmid pTV 18(1) of native bGH containing an extra methionine residue expresses hGH under control of the PL promoter. This at the amino terminus. 4,831,120 7 8 (b) Using the strategy described in (a) but modifying the A2606 and A1563 are derive from SASOO. Their phe structure of the oligodeoxyribonucleotide linker a notypes are SASOO his-ilu_gal+A8(7tCI857AHl class of vectors coding for a series of modi?ed bovine ABAM N+ and SASOO his" ilu~gal+A8 lac ZxA2l growth hormones is constructed. The modi?ed OtCI859 int2 xisl nutL3 AHl), respectively. growth hormones start with methionine at the N-‘ter Preferably the vector is a covalently closed circular minus and are followed by any of the twenty natu double-stranded molecule. However, it is not essential rally occurring amino acids in each of positions 1 and that the vector be covalently closed. 2, and any of the twenty amino acids other than Glu, The vector achieves its enhanced expression levels Gln, Lys, Met or Trp in position 3. Proceeding from after the host cell is heated to a temperature at which position 4 to the COOH-terminus the sequence is the C1 repressor is destroyed. A temperature above identical to that of native bGH. about 42° C. is effective for this purpose and since it is FIG. 15. Tibia test. This ?gure shows the comparison desired that unnecessary heat damage to the host cells between effect of pRec § bGH analog and authentic be avoided to as great an extent as possible, it is gener bGH on the bone plate growth of hypophysectomized ally desirable that the temperature never exceed 42‘’ C. rats. 15 by more than a few degrees. One important component of the vector is the ribo DETAILED DESCRIPTION OF THE somal binding site. Suitable sites are C11 from lambda INVENTION bacteriophage having the sequence: A vector has been developed which enables the achievement of enhanced levels of gene expression and 20 TAAGGAAATACTTACAT polypeptide expression. The vector is a double-stranded ATTCCI I IATGAATGTA; DNA molecule. Upon introduction into a suitable bac terial host cell containing the thermolabile repressor C1 a synthetic oligonucleotide having the sequence: and increasing the temperature of the host to a tempera 25 ture at which the repressor is destroyed, the vector TAAGGAAGTACTTACAT V renders the host cell capable of effecting expression of a A'I'I‘CCTTCATGAATGTA; and desired gene inserted into the vector and production of the major head protein gene of bacteriophage lambda polypeptide encoded by the gene. having the sequence: The vector includes in 5’ to 3’ order the following: a DNA sequence hhich contains the promoter and oper IIIIIIIACGGGAIIIIIIIATG ator PLOL from lambda bacteriophage; AAAAAAATGCCCTAAAAAAATAC. the N utilization site for binding antiterminator N pro tein produced by the host cell; Another component of the vector is the restriction a DNA sequence which contains a ribosomal binding enzyme site for insertion of desired genes into the vec site for rendering the mRNA of the desired gene tor in phase with the ATG initiation codon. Numerous capable of binding to ribosomes within the host cell; such sites may be used. The presently preferred sites are - an ATG initiation codon or a DNA sequence which is BamHl, Sacl and Ndel. converted into an ATG initiation codon upon inser The vector also includes an origin of replication from tion of the desired gene into the vector; and a bacterial plasmid capable of autonomous replication in a restriction enzyme site for inserting the desired gene the host cell. Suitable such origins of replication may be into the vector in phase with the ATG initiation co obtained from a number of sources. Presently preferred don. ~ are origins of replication derived from pBR322 or pRl. The vector also includes a DNA sequence which A DNA sequence which contains a gene associated contains an origin of replication from a bacterial plas with a selectable or identi?able phenotypic trait which mid capable of autonomous replication in the host cell is manifested when the vector is present in the host cell and a DNA sequence which contains a gene associated is also a component of the vector. Suitable genes in with a selectable or identi?able phenotypic trait which clude those associated with temperauure sensitivity or is manifested when the vector is present in the host cell. drug resistance, e.g., resistance to ampicillin, chloram The host for use with the vector is Escherichia coli. phenicol or tetracycline. The presently preferred strains are A1637, A1645, Relative to vectors previously described in the scien A2602 and A1563. All637 is presently the most pre ti?c literature, the vectors of this invention may be used ferred strain. It was obtained from C600 by inserting to obtain enhanced expression of a wide variety of genes transposon containing tetracycline resistance gene encoding desirable polypeptide products. Suitable within the galactose operon as well as the lambda sys genes include those encoding growth hormones, e.g., tem for expression which is close to galactose operon. It bovine, porcine, chicken or human growth hormones; has been deposited with the American Type Culture superoxide dismutase; apoprotein E; viral protein 1 of Collection in Rockville, Maryland, U.S.A. containing foot and mouth disease virus, protein A from S. aureus, various plasmids as described more fully hereinafter. interleukin III, enzymes, or analogs of any of the pre All such deposits were made pursuant to the Budapest ceding. By analog is meant a polypeptide having the Treaty on the International Recognition of the Deposit same activity as the naturally occurring polypeptide but of Microorganisms. having one or more different amino acids at the N-ter All645 was obtained from A1637 by selection for minus of the polypeptide. Gal+ (ability to ferment galactose) as well as loss of The vector may be formed by methods well known tetracycline resistance. It still contains the lambda ex to those skilled in the art to which the invention relates. pression system but part of the transposon has been Such methods are described in greater detail in various removed by selection. Its phenotype is C600 rrm+ publications identi?ed herein, the contents of which are gal+ thr- leu- lac Z‘ (M1857 AHl ABAM N+). hereby incorporated by reference into the present dis