CA1259043A - Method and means for microbial polypeptide expression - Google Patents

Abstract

IMPROVED METHOD AND MEANS FOR MICROBIAL POLYPEPTIDE
EXPRESSION

Abstract of the Disclosure The Specification discloses:
1. Recombinant microbial cloning vehicles com-prising heterologous DNA coding for the expression of mammalian hormone (e.g., somatostatin) and other polypeptides, including plasmids suited for the trans-formation of bacterial hosts. The latter incorporate a regulon homologous to the host in its untransformed state, in reading phase with the structural gene for the heterologous DNA;
2. Cloning vehicles coding for the microbial expression of a protein variously comprising (a) a poly-peptide hapten and additional protein sufficient in size to confer immunogenicity on the product of expression, which may find use in raising antibodies to the hapten for assay use or in the manufacture of vaccines; and (b) a desired polypeptide product and additional protein from which the desired product may be cleaved; and 3. Methods of preparing synthetic structural genes coding for the expression of mammalian polypeptides in microbial cloning systems.

Description

DESCRIPTION

IMPROVED MET~OD AND MEA.~7S
-FOR MICROBIAL POLYPEPTIDE EXPRESSION

Technical Field This invention relates to microbial polypeptide expression.

10 Background Genetic information is encoded on double-stranded deoxyribonucleic acid (~DNA" or "genes") according to the order in which the DNA coding strand presents the characteristic bases of its repeating nucleotide components. "Expression" of the encoded information to form polypeptides involves a two-part process. Accor-ding to the dictates of certain control regions ~ n regulons n ) in the gene, RNA polymerase may be caused to move along the coding strand, forming messenger RNA
tribonucleic acid) in a process called "transcription."
In a subsequent "translation" step the cell's ribosomes in conjunction with transf~r RNA convert the mRNA
"message" into polypeptides. Included in the informa-tion mRNA transcribes from DNA are signals for the start and termination of ribosomal translation, as well as the identity and sequence o~ the amino acids which make up the polypeptide. The DNA coding strand com-prises long sequences of nucleotide ~riplets called , ~L2~

"codons" because the characteristic bases o~ the nucleotides in each triplet or codon enco~e specific bits of information. For example, 3 nucleotides read as ATG (adenine-thymine-guanine) result in an mRNA
signal interpreted as "start translation", while termination codons TAG, TAA and TGA are interpreted ~stop translation~. Between the start and stop codons lie the so-called structural gene, whose codons define the amino acid sequence ultimately translated. That definition proceeds according to the well-established "genetic code" te-g-, J. D. ~atson, Molecular Biology_ of the Gene W.A. Benjamin Inc., N. Y., 3rd ed. 1976) wh-ch describes the codons for the various amino acids.
The genetic code i5 degenerate in the sense that different codons may yield the same amino acid, but precise in that for each amino acid there a~e one or more codons for it and no other. Thus, for example, all of the codons TCT, TCA, TCG, AGT, and A5C, when read as such, encode for serine and no other amino acid.
During translation the proper reading phase or reading frame must be maintained. Consider for example what happens when the ribosome reads different bases as the beginning of a codon ~underlined) in the sequence . . . GCTGGTTGTAAG . . .:
. . .GCT GGT TGT AAG . . . ~ . . .Ala-Gly-Cys-Lys. .
. . .G CTG GTT GTA AG. . . , . ~ .Leu-Val-Val. . .
. . .GC TGG T TAA G. . . ~ . . .Trp-Leu-(STOP).

The polypeptide ultimately produced, then, depends vitally upon the spatial relationship of the structural gene with respect to the regulon.
A clearer understanding of the process of genetic expression will emerge once certain components of genes are defined:
OPeron -- A gene comprising structural gene(s) for polypeptide expression and the control region (~regulon") which regulates that expression.

:.

~L2~0~

Promoter -- A gene within the regulon to which RNA
polymerase must bind for initiation of transcription.
~ _rator -- A gene to which repressor protein may bind, thus preventing RNA polymerase binding on the adjacent promoter.
Inducer -- A substance which deactivates repressor protein, freeing the operator and permitting RNA poly-merase to bind to promoter and commence transcription.
Catabolite Activator Protein (nCAP") Bindi~
-- A gene which binds cyclic adenosine monophosphate (nc AMPn) - mediated CAP, also commonly required for initiation of trancription~ The CAP binding site may in particular cases be unnecessary. For example, a promoter mutation in the lactose operon of the phage ~ plac W 5 eliminates the requirement for cAMP and CAP
expression. J. Beckwith et al, J. Mol. Biol 69, 155-160 (1972).
Promoter-O~erator 5ystem -- As used herein, an operable control region of an operon, with or without respect to its inclusion of a CAP binding site or capacity to code for repressor protein expression.
Further by way of definition, and for use in the discussion of recombinant DNA which follows, we def ine the following:
Cloning Vehicle - Non-chromosomal double stranded DNA comprising an intact "replicon" such that the vehicle is replicated, when placed within a unicellular organism ("microbe") by a process of "transformation".
An organism so transformed is called a 'transformantn.
3~ Plasmid - For present purposes, a cloning vehicle derived from viruses or bacteria, the latter being "bacterial plasmids."
Complementarity -- A property conferred by the base sequences of single strand DNA which permits the formation of double stranded DNA through hydrogen bonding between complementary bases on the respective strands. Adenine (A) complements thymine (T), while guanine (G~ complements cytosine tC).

Advances in biochemistry in recent years have led to the construction of "recombinant" cloning vehicles in which, for example, plasmids are made to contain exogenous DNA. In particular instances the recombinant may include ~heterologous" DNAI by which is meant DNA
that codes for polypeptides ordinarily not produced by the organism susceptible to transformation by the recom-binant vehicle. Thus, plasmids are cleaved to provide linear DNA having ligatable termini. These are bound to an exogenous gene having ligatable termini to provide a biologically functional moiety with an intact replicon and a desired phenotypical propertyO The recombinant moiety is inserted into a microorganism by transforma-tion and transformants are isolated and cloned, with the object of obtaining large populations capable of expres-sing the new genetic information. Methods and means of forming recombinant cloning vehicles and transforming organisms with them have been widely reported in the literature. See, e.g., H. ~. Heynecker et al, Nature 263, 748-752 (1976); Cohen et al, Proc. Nat. Acad. Sci.
VSA 69, 2110 (1972); ibid., 70, 1293 (1973); ibid., 70, 3240 (1973); ibid., 71, 1030 (1974); Morrow et al, Proc.
Nat. Acad. Sci. ~.S.A. 71, 1743 (1974); Novick, Bacteri-oloqical Rev., 33, 210 (1969); Hershfield et al, Proc.
Nat'l. Acad. Sci. U.S.A. _ , 3455 (1974) and Jackson et al, ibid. 69, 2904 (1972). A generalized discussion of the subject appears in S. Cohen, Scientific American 233, 24 (1975).

A variety of techniques are available for DNA
recombination, according to which adjoining ends of separate DNA fragments are tailored in one way or another to facilitate ligation. The latter term refers to the formation of phosphodiester bonds between adjoining nucleotides, most often through the agency of the enzyme T4 DNA ligase. Thus, blunt ends may be directly ligated. Alternatively, fragments containing complementary single s~rands at their adjoining ends are advantaged by hydrogen bonding which positions the respective ends for subsequent ligation. Such single strands, referred to as cohesive termini, may be formed by the addition of nucleotides to blunt ends using ter-minal transferase, and sometimes simply by chewing backone strand of a blunt end with an enzyme such ~ -exonu-clease. Again, and most commonly, resort may be had to restriction endonucleases, which cleave phosphodiester bonds in and around unique sequences of nucleotides of about 4-6 base pairs in length. Many restriction endo-nuclea~es and their recognition sites are known, the so-called Eco RI endonuclease being most widely employed.
Restriction endonucleases which cleave double-stranded DNA at rotationally symmetric "palindromes" leave cohe-sive termini. Thus, a plasmid or other cloningvehicle may be cleaved, leaving termini each comprising half the restriction endonuclease recognition site. A
cleavage product of exogenous DNA obtained with the same restriction endonuclease will have ends complementary to those of the plasmid termini. Alternatively, as dis-closed infra, synthetic DNA comprising cohesive termini may be provided for insertion into the cleaved vehicle.
To discourage rejoinder of the vehicles' cohesive ter-mini pending insertion of exogenous DNA, the terminii can be digested with alkaline phosphatase, providing molecular selection for closures incorporating the exo-genous fragment. Incorporation of a fragment having the proper orientation relative to other aspects of the vehicle may be enhanced when the fragment supplants vehicle DNA excised by two different restriction endo-nucleases, and itself comprises termini respectively constituting half the recognition sequence of the different endonucleases.
Despite wide-ranging work in recent years in recombinant DNA research, few results susceptible to immediate and practical application have emerged. This ~as proven especially so in the case of failed attempts to express polypeptides and the like coded for by "synthetic DNA", whether constructed nucleotide by nucleotide in the conventional fashion or obtained by reverse transcription from isolated in RNA (complemen-tary or ~cDNA~)o In this application we describe what appears to represent the first expression of a func-5 tional polypeptide product from a synthetic gene,together with related developments which promise wide-spread application. The product referred to is somatostatin (Guillemin U.S.P. 3,904,594), an inhibi~or of the secretion of growth hormone, ins~lin and glucagon whose effects suggest its application in the treatment of acromegaly acute pancreatitis and insulin-dependent diabetes. See R. ~uillemin et al, Annual Rev. Med. 27 379 (1976). The somatostatin model clearly demonstrates the applicability of the new developments described here on numerous and beneficial fronts, as will appear from the accompanying drawings and more clearly from the detailed description which follows.

_mmary of Invention According to the invention there is provided a method of producing expression of a heterologous structural gene therefor in a recombinant microbial cloning vehicle, wherein the structural gene is in reading phase with a DNA sequence coding for a protein other than said polypeptide so that expression yields a precursor protein comprising both the amino acid sequence of the polypeptide and additional protein containing a selective cleavage site adjacent the desired polypeptide's amino acid sequence.
Also according to the invention there is provided a recombinant microbial cloning vehicle comprising a regu-lon, a structural gene coding for the amino acid sequence of a desired polypeptide and one or more termination codon(s), wherein a DNA sequence coding for additional protein is interposed between said regulon and termina-tion codon~s~ without altering the reading frame of said structural gene such that a precursor protein comprising both the amino acid se~uence of the desired polypeptide and that of additional protein results from expression, the additional protein comprising a selective cleavage site adjacent the amino acid sequence of the desired polypeptide. Preferably, expression yields a conjugate protein consisting essentially of the amino acid sequences of the hapten and additional protein, the latter being sufficiently large as to confer immunogeni-city on the conjugate.
The invention further relates to a recombinant microbial cloning vehicle comprising a regulon, a DNA
sequence for a polypeptide includ;ng a first amino acid sequence and a second amino acid sequence, and one or more termination codon(s), wherein codons coding for ~he second amino acid sequence are interposed between said regulon and termination codon(s) without altering the reading frame of said DNA sequence such that a polypeptide comprising both the first and second amino acid sequences results from expression, there being a selective cleavage site adjacent the first amino acid sequence.
Also, part of the invention is the production of an immunogenic substance comprising a polypeptide hapten, which includes a) providing a recombinant microbial cloning vehicle containing a heterologous structural gene for the hapten and, in reading phase therewith, ? DNA
se~uence coding for additional protein suffi~ient in size as to render the product of DNA expression immuno-genic; and b) occasioning expression of a conjugate -polypeptide consisting essentially of the amino acid sequence of said Aapten and said additional protein.

-7a-Brief Description of the_Drawin~
The accompanying drawings illustrate one context in which preferred embodiments of the invention find application, i.e~, expression of the hormone somato-statin by bacterial transformants containing recombinantplasmids.
Fig~re 1. Schematic outline of the process: the gene for somatostatin, made by chemical DNA synthesis, is fused to the E. coli ~ -galactosidase gene on the plasmid pBR322. After transformation into E. coli, the recombinant plasmid directs the synthesis of à precursor protein which can be specifically cleaved ~n vitro at methionine residues by cyanogen bromide to yield active mammalian polypeptide hormone. A, T, C and G
denote the characteristic bases (respectively adenine, thymine, cytosine and guanine) of the devxyribonucleo-tides in the coding strand of the somatostatin gene.

. ........... .

:L~5~ 3 Figure 2. Schematic structure of a synthetic gene whose coding strand (i.e., the "upper" strand) comprises codons for the amino acid sequence of somatostatin (given).
Figure 3. Schematic illustration of preferred method for construction of nucleotide trimers used in constructing synthetic genes. In the conventional notation employed to depict nucleotides in Figure 3, the 5' OH is to the left and the 3' O~ to the right, e.g.

5' 1 3' HO ~ OH
Figure 4. Flow chart for the construction of a recombinant plasmid te.g., pSOM11-3) capable of expressing a somatostatin (nSOM")- containing protein, beginning with the parental plasmid pBR322. In Figure 4 the approximate molecular weight of each plasmid is stated in daltons ("d"). Apr and Tcr respectively denote genes for ampicillin and tetracycline resistance, while Tcs denotes tetracycline susceptibility resul-ting from excision of a portion of the ~cr gene. Therelative positions of various restriction endonuclease specific cleavage sites on the plasmids are depicted (e.gO, Eco RI, Bam I, etc.).
Finures 5A and SB. The nucleotide sequences of ~, _ key portions of two plasmids are depicted, as is ~he direction of messenger RNA (~mRNA") transcription, which invariably proceeds from the 5' end of the coding strand. Restriction endonuclease substrate sites are as shown. Each depicted sequence contains both the control elements of the lac ~lactose) operon, and codons for expression of the amino acid sequence of somatostatin (italics). The amino acid sequence numbers for ~-galactosidase ("~ -gal") are in brackets.

-~2~ 3 _9_ Fiy~res 6 8. As more particularly described in the "Experimental" discussion, infra, these depict the results of comparative radioimmune assay experiments which demonstrate the somatostatin activity of product expressed by the recombinant plasmids.
Figure 9. Schematic structure of synthetic genes whose coding strands comprise codons for the amino acid sequences of the A and B strands of human insulin.
Figure 10. Flow chart for construction of a recombinant plasmid capable of expressing the B chain of human insulin.

Detailed Description - 15 1. Preparation of Genes Coding for Heterologous PolyPeptide DNA coding for any polypeptide of known amino acid sequence may be prepared by choosing codons according to the genetic code. For ease in purification, etc., oligodeoxyribonucleotide fragments of, for example, from about 11 to about 16 nucleotides are prepared separately, then assembled in the desired sequence.
Thus, one prepares first and second series of oligode-oxyribonucleotide fragments of convenient size. The first series, when joined in proper sequence, yield a DNA coding strand for polypeptide expression (see, e~g., Figure 2, fragments A, B, C and D). The second series, when likewise joined in proper sequence, yield a strand complementary to the coding strand (e~g., Figure 2, fragments E, F, G and H). The fragments of the respec-tive strands preferably overlap such that complementa-rity promotes their self assembly through hydrogen bonding of the cohesive terminii of fragment blocks.
Following assembly, the structural gene is completed by ligation in the conventional manner.
The degeneracy of the genetic code permits sub-stantial freedom in the choice of codons for any given amino acid sequence. For present purposes, however, 1;2S~ 3 codon choice was advantageously guided by three consi-derations. First, codons and fragments were selected, and fragment assembly was staged, so as to avoid undue complementarity of the fragments, one with another, save for fragments adjacent one another in the intended gene.
Secondly, sequences rich in AT base pairs (e.g., about five or more) are avoided, particularly when preceded by a sequence rich in GC base pairs, to avoid premature termination of transcription. Thirdly, at least a majority of the codons chosen are those preferred in the expression of microbial genomes (see, e.g., W. Fiers, et al, Nature 260, 500 (1976). For purposes of the appended claims, we define the following as codons "preferred for the expression of microbial genomes":
TABLE I
PREFERRED ASSIGNMENT OF CODONS
First Position Second Position Third Position (5' End)~Read Across) (3' End) (Read Down) T C AG (Read Down) phe ~ - cys T
T phe ser tyr --- C
leu --- Stop Stop A
--- ser Stop trp G
leu pro his arg T
leu pro his arg C
leu pro gln --- A
C --- nro aln --- G
ile thr asn --- T
ile thr asn ser C
A
A met(start)thr lys - - G
val ala asp gly T
val --- asp --- C
val - - glu --- A
G val ala ~lu --- G
Most preferably in the case of somatostatin, the amino acid (codon) relationships of the structural gene are: gly ~GGT); cys (TGT); lys (AAG); trp (TGG); ala (GCTr GCG); as- (AAT, AAC); phe (TTC, TTT); thr (ACT, ACG); and ser (TCC, TCG).

Where the structural gene of a desired polypeptide is to be inserted in a cloning vehicle for expression as such, the gene is preceded by a "start" codon (e.g., ATG) and immediat~ly followed by one or more termination or stop codons (see Fig. 2). However, as described nfra, the amino acid sequence of a particular polypeptide may be expressed with additional protein preceding and/or following it. If the intended use of the polypeptide requires cleavage of the additional protein, appropriate cleavage sites are coded for adjacent the polypeptide -additional protein codon junction. Thus, in Figure 1 as an example, the expression product is a precursor protein comprising both somatostatin and the greatest part of the ~ -galactosidase polypeptide. Here ATG is not requ~red to code for the start of translation because ribosom~1 translation of the additional ~ -gal protein reads through into the somatostatin structural gene.
Incorporation of the ATG signal/ however, codes for the production of methionine, an amino acid specifically cleaved by cyanogen bromide, affording a facile method for converting precursor protein into the desired polypeptide .
Figure 2 also exemplifies a further feature pre-ferred in heterologous DNA intended for recombinant employment, i.e.~ the provision of cohesive terminii, preferably comprising one of the two strands of a restriction endonuclease recognition site. For reasons previously discussed, the terminii are preferably designed to create respectively different recognition sites upon recombination.
While the developments described here have been demonstrated as successful with the somatostatin model, it will be appreciated that heterologous DNA coding for virtually any known amino acid sequence may be employed, mutatis mutandis. Thus, the techniques previously and hereafter discussed are applicable, mutatis m~tandis, to the production of poly(amino)acids, such as polyleu-cine and polyalanine; enzymes; serum proteins; analgesic ~25~

polypeptides, such as ~ -endorphins, which modulate thresholds of painl etc. Most preferably, the polypep-tides produced as such will be mammalian hormones or intermediates therefor. Among such hormones may be mentioned, e.g., somatostatin, human insulin, human and bovine growth hormone, luteinizing hormone, ACTH, pancreatic polypeptide, etc. Intermediates include, for example, human preproinsulin, human proinsulin, the A and B chains of human insulin and so on. In addition to DNA made in vitro, the heterologous DNA may comprise cDNA resulting from reverse transcription from mRNA.
See, e.g., Ullrich et al, Science 196, 1313 (1977).

2. Recombinants Coding for the Expression of Precursor Protein In the process schematically depicted in Figure 1, expression yields a precursor protein comprising both a polypeptide coded for by a specific heterologous struc-tural gene ~somatostatin) and additional protein (com-prising a portion of the ~ -galactosidase enzyme). A
selective cleavage site adjacent the somatostatin amino acid sequence permits subsequent separation of the desired polypeptide from superfluous protein. The case illustrated is representative of a large class of proce-dures made available by the techniques described herein.
Most commonly, cleavage will be effected outsidethe replicative environment of the Plasmid or other vehicle as, for example, following harvest of the micro-bial culture. In this fashion temporary conjugation of small polypeptides with superfluous protein may preserve the former against, e.g., in vivo degradation by endoge-nous enzymes. At the same time, the additional protein will ordinarily rob the desired polypeptide of bioacti-vity pending extra-cellular cleavage, with the effect of enhancing the biosafety of the procedure. In parti-cular instances, of course, it may prove desirable to effect cleavage within the cell. For example, cloning vehicles could be provided with DNA coding for enzymes which convert insulin precursors to the active form, operating in tandem with other DMA coding for expression of the precursor form.
In the preferred case, the particular polypeptide desired lacks internal cleavage sites corresponding to that employed to shed superfluous protein, although it will be appreciated that where that condition is not satisfied competition reactions will yet give the desired product, albeit in lower yield. Where the desired product is methionine-free, cyanogen bromide cleavage at methionine adjacent the desired sequence has provén highly effective. Likewise, arginine - and lysine-free products may be enzymaticaily cleaved with, e.g., trypsin at arg-arg, lys-lys or like cleavage sites adjacent the desired sequence. In the case where cleavage leaves, e.g., unwanted arginine attached to desired product, it may be removed by carboxypeptidase digestion. When trypsin is employed to cleave at arg-arg, lysine sites within the desired polypeptide may first be protected, as with maleic or citraconic anhydrides. The cleavage techniques dis-cussed here by way of example are but representative of the many variants which will occur to the art-skilled in light of the specification.
Cleavable protein may be expressed adjacent either the C- or N-terminals of a specific polypeptide, or even within the polypeptide itself, as in the case of the included sequence which distinguishes proinsulin and insulin. Again, the vehicle employed may code for expression of protein comprising repeated sequences of the desired polypeptide, each separated by selective cleavage sites. Most preferably, however, codons for superfluous protein will be translated in advance of the structural gene of the desired product, as in the case illustrated in the Figures. In every case care should be taken to maintain the proper reading frame relative to the regulon.

............. . . .

3 Expression of Immunogens .

The ability to express both a specific polypeptide and superfluous protein provides useful tools for the production of immunogenic substances. Polypeptide ~ 5 ~haptensU (i.e. substances containing determinants specifically bound by antibodies and the like but ~ ordinarily too small to elicit an immune response) can be expressed as conjugates with additional protei~
sufficient in size to confer immunogenicity. Indeed, 1~ the ~ -gal - somatostatin conjugate produced here by way of example is of immunogenic size and may be expected to raise antibodies which bind the somatostatin hapten.
Proteins comprising in excess of 100 amino acids, most commonly in excess of 200 such, exhibit immunogenic character.
Conjugates prepared in the foregoing fashion may be employed to raise antibodies useful in radioimmune or other assays for the hapten, and alternatively in the production of vaccines. We next describe an example of the latter application. Cyanogen bromide -or other cleavage products of viral coat protein will yield oligopeptides which bind to antibody raised to the protein itself. Given the amino acid sequence of such an oligopeptide hapten, heterologous DNA therefore may be expressed as a conjugate with additional protein which confers immunogenicity. Use of such conjugates as vaccines could be expected to diminish side reactions which accompany use of coat protein itself to confer immunity.

4 The Control Elements Figure 1 depicts a process wherein a transformant organism expresses polypeptide product from heterologous DNA brought under the control of a regulon "homologousn to the organism in its untransformed state. Thus, lactose-dependent E. Coli. chromosomal DNA comprises a lactose or nlac" operon which mediates lactose digestion by, inter alia, elaborating the enzyme ~-galactosidase.

~25i~ 3 In the particular instance illustrated, the lac control elements are obtained from a bacteriophage, ~ plac 5, which is infective for the E. Coli. The phage's lac operon, in turn, was derived by transduction from the same bacterial species, hence the "homology". Homolo-gous regulons suitable for use in the disclosed process may alternatively deriv~ from plasmidic DNA native to the organismA
The simplicity and efficiency of the lac promoter-operator system commend its use in the systems wedescribe, as does its ability to be induced by IPTG
(isopropylthio- ~ -D galactoside). Of course, other operons or portions thereof could be employed as well, e.g., lambda promoter-operatort arabinose operon (phi 80 dara), or the colicine El, galactose, alkaline phosphatase or tryptophan operons. Promoter-operators derived from the latter (i.e.t "tryp operon") would be expected to confer 100~ repression pending induction (with indoleacrylic acid) and harvest.

5~ Plasmid Construction Generally The details of the process schematically illustra-ted in Figure 4 appear from the Experimental sectiont infra. At this pointt howevert it is useful to briefly discuss various of the techniques employed in construc-ting the recombinant plasmid of the preferred embodiment.
The cloning and expression of the synthetic soma-tostatin gene employed two plasmids. Each plasmid has an EcoRI substrate site at a different region of the ~ -galactosidase structural gene (see Figures 4 and 5).
The insertion of the synthetic somatostatin DNA fragment into the EcoRI sites of these plasmids brings the expression of the genetic information in that fragment under control of the lac operon controlling elements~
Following the insertion of the somatostatin fragment into these plasmids, translation should result in a somatostatin polypeptide preceded either by 10 amino acid (pSOM1) or by virtually the whole ~ -galactosidase subunit structure (pSOM11-3).
The plasmid construction scheme initiates with plasmid pBR322, a well-characterized cloning vehicle.
Introduction of the lac elements to this plasmid was accomplished by insertion of a ~aeIII restriction endonuclease fragment (203 nucleotides) carrying the lac promoter, CAP binding site, operator, ribosome binding site, and the first 7 amino acid codons of the ~ -galactosidase structural gene. The HaeIII fragment was derived from ~ plac5 DNA. The EcoRI-cleaved PBR322 plasmid, which had its termini repaired with T4 DNA
polymerase and deoxyribonucleotide triphosphates, was blunt-end ligated to the HaeIII fragment to create EcoRI termini at the insertion points. Joining of these HaeIII and repaired EcoRI termini generate the EcoRI restriction site (see Fig. 4 and 5) at each terminus. Transformants of E. Coli RR1 with this DNA
were selected for resistance to tetracycline (Tc) and ampicillin (Ap) on 5-bromo-4-chloro-indoylgalactoside (X-gal) mediu~. On this indicator medium, colonies constitutive for the synthesis of ~ -galactosidase, by virtue of the increased number of lac operators titra-ting repressor, are identified by their blue color.
Two orientations of the HaeIII fragment are possible but these were distinguished by the asymmetric location of an Hha restriction site in the fragment. Plasmid pBH10 was further modified to eliminate the EcoRI
endonuclease site distal to the lac operator (pBH20).
The eight chemically synthesized oligodeoxyribo-nucleotides (Fig. 2) were labeled at the 5' termini with [32p]_ -ATP by polynucleotide kinase and joined with T4 DNA ligase. Through hydrogen bonding b~tween the overlapping frayments, the somatos~atin gene self-assembles and eventually polymerizes into larger molecules because of the cohesive restriction site termini. The ligated products were treated with EcoRI

and BamHI restriction endonucleases to generate the somatostatin gene as depicted in Figure 2.
The synthetic ssmatostatln gene fragment with EcoRI and BamHI termini was ligated to the pBH20 plasmid, previously treated with the EcoRI and BamHI restriction endonucleases and alkaline phosphatase. The treatment with alkaline phosphatase provides a molecular selection for plasmids carrying the inserted fragment. Ampicillin-resistant transformants obtained with this ligated DNA
were screened for tetracycline sensitivity and several were examined for the insertion of an EcoRI-Bam~I
fragment of the appropriate size.
Both strands of the EcoRI-Bam~I fragments of plasmids from two clones were analyzed by nucleotide sequence analysis starting from the BamHI and EcoRI
sites. The sequence analysis was extended into the lac controlling elements; the lac fragment sequence was intact, and in one case, pSOMl, the nucleotide sequence of both strands were independently determined each giving the sequence depicted in Figure 5A.
The EcoRI-Pst fragment of the pSOMl plasmid, with the lac-controlling element, was removed and replaced with the EcoRI-Pst fragment of pBR322 to produce the plasmid pSOMll. The EcoRI fragment of plac 5, carry-ing the lac operon control region and most of the ~ -galactosidase structural gene, was inserted into the EcoRI site of pSOM11. Two orientations of the EcoRI
lac fragment of ~plac 5 were expected. One of these orientations would maintain the proper reading frame into the somatostatin gene, the other would not. Analy sis of independently isolated clones for somatostatin activity then identified clones containing the properly oriented gene, of which the clone designated pSOM11-3 was one.

6. The Micr~
Various unicellular microorganisms have been pro-posed as candidates for transformation, such as bacteria, , ~S~3 fungii and algae. That is, those unicellular organisms which are capable of being grown in cultures or fermen-tation. Bacteria are for the most part the most conve-nient organisms to work with. Bacteria which are sus-ceptible to transformation include members of the Enter-obacteriaceae, such as strains of Escherichia coli and Salmonella; Bacillaceae, such as Bacillus subtillis;
Pneumococcus; Strep~ococcus, and Haemophilus influenzae.
The particular organism chosen for the somatostatin work next discussed was E.Coli. strain RRl, genotype:
Pro~Leu~Thi~RB~MB rec A+ Strr Lac y~ E. Coli.
RRl is derived from E. Coli. HB101 (H.W. Boyer, et al, J. Mol. Biol. (1969) 41, 459-472) Dy mating with E. Coli K12 strain KL16 as the Hfr donor. See J. H. Miller, Experiements in Molecular Genetics ~Cold Spring Harbor, New York, lg72). Cultures of both E. Coli RRl and E.
Coli. RRl ~pBR322) have been deposited with the American Type Culture Collection without restriction as to access, respectively ATCC Nos. 31343 and 31344. The somatosta-tin-producing organism has likewise been deposited ~ATCC No. 31446].
.
In the case of human insulin, A and B chain genes were cloned in E Coli K-12 strain 294 (end A, thi-, hsr-, hsmk~), ATCC No. 31447 , and that organism employed in expression of the A chain ( E Coli K-12 strain 294 [pIAl], ATCC No. 31448 ). The B chain of human insulin was first expressed in a derivative of HB101, i.e., E. coli K-12 strain D1210 a lac+ (iQo+zty+)t and that B yene-containing organism has likewise been deposited (ATCC No. 31449). Alternatively, the B gene may be inserted in and expressed from the organism first mentioned, i.e., strain 294.

EXPERIMENTAL

I SOMATOSTATIN

1. Construction of Somatostatin Gene Fragments Eight oligodeoxyribonucleotides respectively labeled A through H in Figure 2 were first constructed, principally by the modified triester method of K.
Itakura et al, J. Am. Chem. Soc. 97, 7327 (1975).
However, in the case of fragments C, E and ~ resort was had to an improved technique in which fully protected trimers are first prepared as basic units for building longer oligodeoxyribonucleotides.
The improved technique is schematically depicted in Figure 3, wherein B is thymine, N-benzoylated adenine, N-benzoylated cytosine or N-isobutyrulated g~anine. In brief, and with reference to Figure 3, with an excess of I (5 mmole), the coupling reaction with II (1 mmole) went almost to completion in 60 min with the aid of a powerful coupling reagent, 2,4,6-triisopropylbenzene-sulfonyl tetrazolide (TPSTe, 4 mmole; 2). After removal of the 5'- protecting qrou~ with 2% benzene sulfonic acid solution, the S'-hydroxyl dimer V could be separated from an excess of 3'-phosphodiester monomer IV by simple solvent extraction with aqueous NaHC03 solution in CHC13. The fully protected trimer block was prepared successively from the 5'-hydroxyl dimer V, I (2 mmole), and TPSTe (4 mmole) and isolated by chromat-ography on silica gel, as in B.T. Hunt et alc Chem. and Ind 1967,_1868 (1967). The yields of trimers made according to the improved technique appea~ from Table II
The eight oligodeoxyribonucleotides, after removal of all protecting groups, were purified by high-pressure liquid chromatography on Permaphase AAX ~ A. Henry et al J. Chrom. Sci. II, 3~8 (1973)). The purity of each oligomer was checked by homochromatography on thin-layer DEAE-cellulose and also by gel electrophoresis in 20%

. . .

~L;2~ 3 acrylamide slab after labeling of the oligomers with [ ~ -32P]-ATP in the presence of polynucleotide kinase.
One major labeled product wa~ obtained from each DNA
fragment.
TABLE II
Yields of Full~ Protected Trimers SequenceYield SequenceYield TTT 81% ATG 69 TTT 75% GCC 61%
GGA 41% CCA 72%
AGA 49% CAA 72%
ATC 71% TTA 71%
CCT 61% CAT 52%
ACA 63% CCC 73 ACC 65~ AAC 59 CGT 51% GAT 60%

2. Ligation and Acr~lamide Gel Analysis of Somatostatin DNA
The 5' OH termini of the chemically synthesized fragments A through H were separately phosphorylated with T4 polynucleotide kinase. [32p]_ ~-ATP was used in phosphorylation so that reaction products could be monitored autoradiographically, although it will be appreciated that unlabelled ATP would serve as well were autoradiography dispensed with. Just prior to the kinase reaction, 25 c of 1 ~-32P]ATP (approx.
~5 1500 c/mMol) (Maxam and Gilbert, Proc. Nat Acad~ Sci.
U.S.A. 74, 1507 (1977) was evaporated to dryness in 0.5 ml Eppendorf tubes. Five micrograms of fragment were incubated with 2 units of T4 DNA kinase (hydroxylapatite fraction, 2500 units/ml ), in 70 mM Tris-HCl pH 7.6, 10 mM MgC12, 5 mM dithiothreitol in a total volume of 150 ~1 for 20 min at 37C. To insure maximum phosphory-lation of the fragments for ligation purposes, 10 ~1 of a mixture consisting of 70 mM Tris HCl pH 7.6, 10mM
MgC12, 5 mM dithiothreitol, 0.5 mM ATP and two units 3~ of DNA kinase were added and incubation continued for an additional 20 min at 7C. The frayments (250 ng/~l) ~rs~

were stored at -20C without further treatment.
Rinased fragments A, B, E, and F (1.25 ~g each) were ligated in a total volume of 50 ~1 in 20 mM Tris-HCl pH

7.6, 10 mM Mg C12, 10 mM dithiothreitol, 0.5 mM ATP
and 2 units of T4 DNA ligase (hydroxylapatite f~action, 400 units/ml; 27), for 16 hr at 4C. Fragments C, D, G
and H were ligated under similar conditions. Samples of 2 ~1 were removed for analysis by electrophoresis on a 10% polyacrylamide gel followed by autoradiography (H. L. Heyneker et al, Nature 263, 748 (1976)) in whtch unreacted DNA fragments are represented by fast migrating material and wherein the monomeric form of the ligated fragments migrate with bromophenol blue dye (BPB).
Some dimerization also occurs by reason of the cohesive ends of the ligated fragments A, B, E and F, and of the ligated fragments C, D, G and H. These dimers represent the slowest migrating material, and may be cleaved by restriction endonuclease EcoRI and BamHI, respectively.
The two half molecules (ligated A + 8 + E + F and ligated C + D + G ~ H) were joined by an additional ligation step carried out in a final volume of 150 ~1 at 4C for 16 hr. One microliter was removed for analysis. The reaction mixture was heated for 15 min at 65C to inactivate the T4 DNA ligase. The heat treatment does not affect the migration pattern of the DNA mixture. Enough restriction endonuclease BamHI was added to the reaction mixture to cleave the multimeric forms of the somatostatin DNA in 30 min at 37C. After the addition of NaCl to 100 mM, the DNA was digested with EcoRI endonuclease. The restriction endonuclease digestions were terminated by phenol-chloroform extrac-tion of the DNA. The somatostatin DNA fragment was purified from unreacted and partially ligated DNA frag-ments by preparative electrophosresis on a 10% polyarry-lamide gel. The band containing the somatostatin DNAfragment was excised from the gel and the DNA was eluted ~y slicing the gel into small pieces and extracting the DNA with elution buffer (0.5 M ammonium acetate, 10 mM
~ .~

-2~-MgC12, 0.1 mM EDTA, 0.1% SDS) overnight at 65~C. The DNA was precipitated with 2 volumes of ethanol, centri-fuged, redissolved in 200 ~1 10 mM Tris-HCl pH 7.6 and dialyzed against the same buffer resulting in a somato-statin DNA concentration of 4 ~g/ml~

3. Construction of Recombinant Plasmids Figure 4 schematically depicts the manner in which recombinant plasmids comprising the somatostatin gene were constructed, and may be referred to in connection with the following more particularized discussion.

A. The Parental Plasmid pBR322 The plasmid chosen for experimental somatostatin cloning was pBR322, a small (molecular wt. approx. 2.6 megadaltons) plasmid carrying resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). As indicated in Figure 4, the ampicillin resistance gene includes a cleavage site for the restriction endonu-clease P I, the tetracycline resistance gene includes a similar site for restriction endonuclease BamHI, and an EcoRI site is situated between the Apr and TCr genes. The plasmid pBR322 is derived from pBR313, a 5.8 megadalton AprTcrColimm plasmid (R.L. Rodriquez et al, ICN-UCLA Symposia on Molecular and Cellular Biology 5, 471-77 (1976), R. L. Rodriquez et al, Con-struction and Characterization of Cloning Vehicles, in Molecular Mechanisms in the Control of Gene Expression, pp. 471-77, Academic Press, Inc. (1976~. Plasmid pBR
322 is characterized and the manner of its derivation fully described in F. Bolivar et al, "Construction and Characterization of New Cloning Vehicles II. A
Multipurpose Cloning System", Gene (November 1977).
B. Construction of Plasmid pBH10 Five micrograms of plasmid pBR322 DNA was digested with 10 units of the restriction endonuclease EcoRI in 100 mM Tr is-HCl pH 7.6, lOOmM NaCl, 6 mM MgC12 at 37C for 30 min. The reaction was terminated by phenol-chloroform extraction; the DNA was then precipitated with two and a half volumes of ethanol and re~uspended in 50 ~1 of T4 DNA polymerase buffPr t67 mM Tris-HCl pH

8.8, 6.7 mM MgC12, 16.6 mM (~H~)2SO4, 167 ~g/ml bavine serum albumin, 50 ~M of each of the dNTP's; A. Panet et al, Biochem. 12, 5045 (1973). The reaction was started by the addition of 2 units of T4 DNA polymerase. After incubation for 30 min at 37 the reaction was terminated by a phenol-chloroform extraction of the DNA followed by precipitation with ethanol. Three micrograms of plac5 DNA (Shapiro et al Nature 224. 768 (1969)) was digested for 1 hr at 37C with the restriction enzym~ HaeIII (3 units) in 6 mM Tris-HCl pH 7.6, 6 mM
MgC12, 6 mM ~-mercaptoethanol in a final volume of 20 ~1. The reaction was stopped by heating for 10 min at 65C. The pBR322 treated DNA was mixed with the HaeIII
digested ~ plac5 DNA and blunt-end ligated in a final volume of 30 ~1 with 1.2 units of T4 DNA ligase (hydroxylapatite fraction; A. Panet et al, supra) in 20 mM Tris-HCl pH 7.6, 10 mM MgC12, 10 mM dithiothrei-tol, 0.5 mM ATP for 12 hrs at 12C. The ligated DNA
mixture was dialyzed against 10 mM Tris -HCl pH 7.6, and used for transformation of E. coli strain RRl.
Transformants were selected for tetracycline and ampici-llin resistance on minimal medium~ plates containing 40 Jug/ml of 5-bromo-4-chloro-colylgalactoside (X-gal) medium (J.H. Miller, Experiments in Molecular Genetics (Cold Spring Harbor, New York, 1972)). Colonies constitutive for the synthesis of ~-galactosidase were identified by their blue color. After screening 45 independently isolated blue colonies, three of them were found to contain plasmid DNA carrying two EcoRI
sites separated by approximately 200 base pairs. The position of an asymmetrically located HhaI fragment in 'he 203 b.p. HaeIII lac control fragment (W. Gilbert et al, in Protein-Ligand Interactions, ~. Sand and G.

Blauer, Eds. (De Gruyter, Berlin, tl975) pp. 193-210) allows for the determination of the orientation o the ~aeIII fragment, now an EcoRI fragment, in these plasmids. Plasmid pB~10 was shown to carry the fragment in the desired orientation, i.e., lac transcription going into the Tcr gene of the plasmid.

C. Construction of Plasmid pBH20 Plasmid pBH10 was next modified to eliminate the EcoRI site distal to the lac operator. This was accomplished by preferential EcoRI endonuclease cleavage at the distal site involving partial protection by ~NA polymerase of the other EcoRI site localized between the Tcr and lac promoters, which are only about 40 base pairs apart. After binding RNA polymerase, the DNA (5 ~g) was digested with ~coRI (1 unit) in a final volume of 10 pl for 10 min at 37C. The reaction was stopped by heating at 65~C for 10 min. The EcoRI
cohesive termini were digested with Sl nuclease in 25 mM Na-acetate pH 4.5, 300 mM NaCl, lmM ZnC12 at 25C
for 5 min. The reaction mixture was stopped by the addition of EDTA (10 mM final) and Tris-~Cl pH 8 (50 mM
final). The DNA was phenol-chloroform extrac~ed, ethanol precip~tated and resuspended in 100 1 of T4 D~A
ligation buffer. T4 DNA ligase (1 1) was added and the mixture incubated at l2C for 12 hr. The ligated DNA was transformed in E. coli strain RRl, and AprTcr transformants were selected on X-gal-antibiotic medium.
Restriction enzyme analysis of DNA screened from 10 isolated blue colonies revealed that these clones carried plasmid DNA with one EcoRI site. Seven of these colonies had retained the EcoRI site located between the lac and Tcr promotors. The nucleotide sequence from the EcoRI site into the lac-control region of one of these plasmids, pBH20, was confirmed. This plasmid was next used to clone the soma~ostatin gene.

~L2~ 3 D. Construction of Plasmid pSOM 1 Twenty micrograms of the plasmid pBH20 was digested to completion with restriction endonucleases EcoRI and BamHI in a final volume of 50 yl. Bacterial alkaline phosphatase was added (0.1 unit of Worthington BAPF) and incubation was continued for 10 min at 65C. The reactions were terminated by phenol-chloroform extrac-tion and the DNA was precipitated with 2 volumes of ethanol, centrifuged and dissolved in 50 ~1 10 mM
Tris-HCl p~ 7.6, 1 mM EDTA. The alkaline phosphatase treatment effectively prevents self-ligation of the EcoRI, BamHI trea~ed pB~20 DNA, ~ut circular recombinant plasmids containing somatostatin DNA can still be formed upon ligation. Since E. coli RRl is transformed with very low efficiency by linear plasmid DNA, the majority of the transformants will contain recombinant plasmids. Fifty microliters of somatostatin DNA (4 ~g/ml) were ligated with 25 jul of the Bam~I, EcoRI, alkaline phosphatase-treated pB~20 DNA in a totsl volume of 50 yl containing 20 mM Tris-HCl pH 7.6, 10 mM
MgC12, 10 mM dithiothreitol, 0.5 mM ATP, and 4 units of T4 DNA ligase at 22C. After 10, 20 and 30 min, additional somatostatin DNA (40 ng) was added to the reaction mixture (the gradual addition of somatostatin DNA may favor liyation to the plasmid over self-ligation). Ligation was continued for 1 hr followed by dialysis of the mixture against 10mM Tris-HC1 pH
7.6. In a control experiment, BamHI, EcoRI, alkaline phosphatase-treated pBH20 DNA was ligated in the absence of somatostatin DNA under similar conditions.
Both preparations were used without further treatment to transform E. coli RRl. The transformation experiments were carried out in a P3 physical containment facility.
(National Institutes of Health, U.S.A., Recombinant DNA
Reasearch Guidelines, 1976)~ Transformants were selected on minimal medium plates containing 20 ~g/ml Ap and 40 ~g/ml X-gal. Ten transformants, which were all sensitive to Tc, were isolated. For reference these were designated pSOMl, pSOM2, etc. . .pSOM10. In the control experiment no transformants were obtained.
Four out of the ten transformants contained plasmids with both an EcoRI site and BamHI site. The size of the small EcoRI, BamHI fragment of these recombinant plasmids was in all four instances similar to the size of the in vitro prepared somatostatin DNA. Base sequence analysis according to Maxam and Gilbert Proc._ Nat. Acad. Sci. U.S.A. 74, 560 (1977), revealed that the plasmid pSOMl had the desired somatostatin DNA
fragment inserted.
The DNA sequence analysis of the clone carrying plasmid pSOMl predicts that it should produce a peptide comprising somatostatin. However no somatostatin radioimmune activity has been detected in extracts of cell pellets or culture supernatants, nor is the presence of somatostatin detected when the growing culture is added directly to 70% formi~ acid and cyanogen bromide. E. coli. RRl extracts have been observed to degrade exogenous somatostatin very rapidly.
The absence of somatostatin activity in clones carryiny plasmid pSOM 1 could well result from intracellular degradation by endogenous proteolytic enzymes. Plasmid pSOM 1 was accordingly employed to construct a plasmid coding for a precursor protein comprising somatostatin and sufficiently large as to be expected to resist proteolytic degradation.

E. The Construction of Plasmids pSOM 11 and pSOM 11-3 A plasmid was constructed in which the somatosta-tin ~ene could be located at the C-terminus of the ~-galactosidase gene, keeping the translation in phase.
The presence of an EcoRI site near the C-terminus of this gene and the available amino acid sequence of this protein (B. Polisky et al, Proc. Nat. Acad. Sci U.S.A.
73, 3900 (1976), A. V. Fowler et al, Id. at 74, 1507 (1976), A. I. Bukhari et al, Nature New Biolo~ 243, 238 (1973) and K. E. Langley, J. Biol. Chem._250, 2587 (1975)) permitted insertion of the EcoRI BamHi somato-statin gene into the EcoRI site while maintaining the proper reading frame. For the construction of such a plasmid, pSOMl DNA (50 ~g) was digested with the restriction enzymes EcoRI and PstI in a final volume of 100 ul. A preparative 5% polyacrylamide gel was used to separate the large Pst-EcoRI fragment that carrie~ the somatostatin gene from the small fragment carrying the lac control elements. The large band was excised from the gel and the DNA eluted by slicing the gel into small pieces and extracting the DNA at 65C overnight.
In a similar way plasmid pBR322 DNA (50 ~9) was digested with PstI and EcoRI restriction endonucleases and the two resulting DNA fragments purified by preparative electrophoresis on a 5~ polyacrylamide gel. The small PstI-EcoRI fragment from pBR322 (1 yg) was ligated with the large PstI-EcoRI DNA fragment (5Jug) from pSOMl in a final volume of 50 ~1 with 1 unit of T4 DNA ligase at 12C for 12 hrs. The ligated mixture was used to trans-form E. coli RRl, and transformants were selected for ampicillin resistance on X-gal medium. As expected, almost all the Apr transformants (95~) gave white colonies tno lac op~rator) on X-gal indicator plates.
The resulting plasmid, pSOMll, was used in the construc-tion of plasmid pSOM11-3. A mixture of 5 yg of pSOMll DNA and 5 ~g of ~ placS DNA was digested with EcoRI
(10 units for 30 min at 37C). The restriction endonu-clease digestion was terminated'by phenol chloroform extraction. The DNA was then ethanol-precipitated and resuspended in T4 DNA ligase buffer (50 ~1). T4 DNA
ligase (1 unit) was added to the mixture and incubated at 12C for 12 hrs. The ligated mixture was di31yzed agains~ 10 mM Tris-~Cl pH 7.6 and used to ~ransform E.
C _ strain RRl. Transformants were selected for Apr on X-gal plates containing ampicillin and screened for constitutive ~ -galactosidase production. Approximately 2~ of the colonies were blue (pSOMll-l, 11-2 etc.).

Restriction enzyme analysis of plasmid DNA obtained from these clones revealed that all the plasmids carried a new EcoRI fragment of approximately 4.4 megadaltons, which carries the lac operon control sites and most of the ~ -galactosidase gene. Because two orientations of the EcoRI fragment are possible, the asymmetric location of a HindIII restriction site was used to determine which of these colonies were carrying ~his EcoRI frag-ment with lac transcription proceeding into the somato-statin gene. ~indIII-BamHI double digestions indicated that only the clones carrying plasmids pSOM11-3, pSOM11-5, pSOM11-6 and pSOM11-7 contained the EcoRI
fragment in this orientation.

4. Radioimmune Assay for Somatostatin Activit~
The standard radioimmune assays (RIA) for soma-tostatin (A. Arimura et al, Proc Soc. Exp. Biol. Med.
148, 784 (1975)) were modified by decreasing the assay volume and using phosphate buffer. Tyrl1 somatostatin was iodinated using a chloramine T procedure. (Id.) To assay for somatostatin, the sample, usually in 70%
formic acid containing 5 mg/ml of cyanogen bromide was dried in a conical polypropylene tube (0.7 ml, Sarstedt) over moist KO~ under vacuum. Twenty microliters of PBSA
buffer (75 mM NaCl; 75 mM sodium phosphate, pH 7.2; 1 mg/ml bovine serum albumin; and 0.2 mg/ml sodium azide) was added, followed by 40 ~1 of a [125I] somatostatin "cocktail" and 20 ~1 of a 1,000-fold dilution in PBSA
of rabbit antisomatostatin immune serum S39 (Vale et al, 3~ Metabolism 25, 1491 (1976). The [125I] somatostatin cocktail contained per ml of PBSA buffer: 250 pg normal rabbit gamma globulin (Ant*ibodies, Inc.), 1500 units protease inhibitor ("Trasylol", Calbiochem) and about 100,000 counts of ~125I] Tyrll - somatostatin. ~fter at least 16 hour at room temperature, 0~333 ml of goat anti-rabbit gamma globul~n (Antibodies, Inc., P=0.03) in PBSA buffer was added to the sample tubes. The mixture was incubated 2 hr at 37C, cooled to 5C, then *Trade Mark ~2S~ [)43 centrifuged at 10,000 X g for 5 min. The supernatant was removed and the pellet counted in a gamma counter.
With the amount of antiserum used, 20% of the counts was precipitated with no unlabeled competing somatosta-tin. The background with infinite somatostatin (200ng) was usually 3%. One-half maximum competition was obtained with 10 pg of somatostatin. Initial experi-ments with extracts of E. Coli strain RRl ~the recipient strain) indicated that less than 10 pg of somatosta in could easily be detected in the presence of 16 ~g or more of cyanogen bromide-treated bacterial protein.
More than 2 ~9 of protein from formic acid-treated bacterial extracts interfered somewhat by increasing the background, but cyanogen bromide cleavage greatly reduced this interference. Reconstruction experiments showed that somatotatin is stable in cyanogen bromide-treated extracts.

A. Competition by Bacterial_Extracts Strains E. Coli RRl (pSOM11-5) and E. Coli RRl (pSOM11-4) were grown at 37C to 5 x 10~ cells/ml in Luria broth. Then IPTG was added to lmM and growth continued for 2 hr. One-milliliter aliquots were cen-trifuged for a few seconds in an Eppendorf centrifuge and the pellets were suspended in 500~ul of ~0% formic acid containing 5 mg/ml cy~nogen bromide. After approx-imately ~4 hr at room temperature, aliquots were diluted tenfold in water and the volumes indicated in Figure 6 were assayed in triplicate for somatostatin. In Figure 6 ~B/Boll is the ratio of [125I] somatostatin bound in the presence of sample to ~hat bound in the absence of competing somatostatin. Each point is the average of triplicate tubes. The protein content of the undiluted samples was determined to b~ 2.2 mg/ml for E. Coli RRl (pSOM11-5) and 1.5 mg/ml for E. Coli RRl (pSOM11-4).

~2~3 . The Initial Screening of SOMll Glones for Somatostatin :~ .
Cyanogen bromide-treated extracts of 11 clones (pSOM11-2, pSOM11-3, etc.) were made as described above ~or the case of Figure 6. Thirty microliters of each extract was taken in triplicate for radioimmune assay, whose results appear from Fi~ure 7. The range of assay points is indicated. The values for picograms somatostatin were read from a standard curve obtained as part of the same experiment.
* * *
The radioimmune assay results described ~hus far may be summarized as follows. In contrast to the re-sults of experiments with pSOMl, four clones (pSOM11-3 11-5, 11-&, and 11-7) were found to have easily detec-table somatostatin radioimmune activity as appears from Figures 6 and 7. Restriction fragment analysis revealed that pSOM11-3, pSOM11-5, pSOM11-6 and pSOM11-7 had the desired orientation of the lac operon, whereas pSOM11-2 and 11-4 had the opposite orientation. Thus there is a perfect correlation between the correct orientation of the lac operon and the production of somatostatin radioimmune activity.
5 C. Effects of IP~G Induction_and CNBr Cleavaqe on Positive and Negative Clones The design of the somatostatin plasmid predicts that the synthesis of somatostatin would be under the control of the lac operon. The lac repressor gene is not included in the plasmid and the recipient strain (E. coli RRl) contains the wild type chromosomal lac repressor gene which produces only 10 to 20 repressor molecules per cell. The plasmid copy number (and therefore the number of lac operators) is approximately 20-30 per cell, so complete repression is impossible.
As shown in Table III, _ fra the specific activity oE
somatostatin in E. coli RRl (pSOMll-3) was increased by IPTG, an inducer of the lac operon. As expect~d, the level of induction was low, varying from 2.4 to 7 fold.
In experiment 7 (Table III) ~ activity, a measure of the first 92 amino acids of ~ -galactosidase, also was induced by a factor of two~ In several experiments no detectable somatostatin radioimmune activity can be detected prior to cyanogen bromide cleavage of the total cellular protein. Since the antiserum u~ed in the radioimmune assay, S 39, requires a free N-terminal al~nine, no activity was expected prior to cyanogen bromide cleavage.
TABLE III
Somatostatin Radioimmune Specific Activity Abbreviations: Luria Brot~, L8; isopropylthiogalactoside, IPTG; cyanogen bromide, CNBr; somatostatin, SS.
Protein was measured by the method of Bradford, ~nal.
Biochem._72, 248 (1976).
Experiment IPTG CNBr pg SS/ug Number _ train Medium 1 mM 5 mg/ml protein 1 11-2 LB + + ~ 0.1 11-3 LB + + 12 11-4 LB ~ + <0.4 11-5 LB + + 15 2 11-3 LB + + 12 11-3 LB + - <0.1 3 11-3 LB + + 61 11-3 LB - ~ 8 11-3 LB + - cO.l 4 11-3 LB + ~ 71 11-3 VB + glycerol* + + 62 11-3 LB + glycerol + + 250 6 11-3 LB + + 350 11-2 LB + + ~0.1 7 11-3 LB + + 24 11-3 LB _ - + _ 10 *Vogel-Bonner minimal medium plus glycerol.

* trade mark.

~L~5~4~

D. Gel Filtration of Cyanogen Bromide - Treated Extracts Formic acid and cyanogen-treated extracts of the positive clones (pSOM 11-3, 11-5, 11-6, and 11-7) were pooled (Total volume 250 ~1), dried, and resuspended in 0.1 ml of 50% acetic acid. [3H~ leucine was added and the sample was applied to an 0.7 X 47 cm column of Sephadex G-50 in 50% acetic acid. Fifty-microliter aliqusts of the column fractions ~ere assayed for somatostatin. Pooled negative clone extracts (11-2, 11-4, and 11-11) were treated identically. The results appear from Figure 8. On the same column known somatostatin (Beckman Corp.) elutes as indicated (SS).
In this system, somatostatin is well-separated from excluded large peptides and fully included small molecules. Only extracts of clones positive for somatostatin exhibited radioimmune activity in the column fractions and this activity elutes in the same position as chemically synthesized somatostatin.
SUMMARY OF ACTI_ITY INFORMATION

The data establishing the synthesis of a a poly-~eptide containing the somatostatin amino acid sequence are summarized as follows: (1) Somatostatin radioimmune activity is present in E. coli cells having the plasmid pSOM11-3, which contains a somatostatin gene of proven correct sequence and has the correct orientation of the lac EcoRI DNA fragment. Cells with the related plasmid pSOM11-2, which has the same somatostatin gene but an opposite orientation of the lac EcoRI fragment, produce no detectable somatostatin activity; (2) As predicted by the design scheme, no detectable somatostatin radio-immune activity is ob~erved until after cyanogen bro-35- mide treatment of the cell extract; (3) The somatostatin activity is under control of the lac operon as eYidenced by induction by IPTG, an inducer of the lac operon; (4) The somatostatin activity co-chromatographs with known * Trademark .

~2~ 3 somatostatin on Sephadex G-50; (5) The DNA sequence of the cloned somatostatin gene is correct. If translation is out of phase, a peptide will be made which is different from somatostatin at every position. Radio-immune activity is detected indicating that a peptideclosely related to somatostatin is made, and translation must be in phase. Since translation occurs in phase t the genetic code dictates that a peptide with the exact sequence of somatostatin is made; (6) Finally, the above samples of E. coli RRl (pSOM11-3) extract inhibit the release of growth hormone from rat pituitary cells, whereas samples of E. coli RRl (pSOM11-2) prepared in parallel and with identical protein concentration have no effect on growth hormone releaseD
STABILITY, YIELD AND PURIFICATION
OF SOMATOSTA~IN

The strains ca~rying the EcoRI lac operon fragment (pSOM11-2, pSOM11-3, etc.) segregate with respect to the plasmid phenotype. For example, after about 15 generations, about one-half of the E coli RRl (pSOM11-3) culture was constitutive for ~ -galactosidase, i.e., carried the lac opera~or, and of these about half were ampicillin resistant. Strains positive (pSOM11-3) and negative (pSOM11-2) for somatostatin are ~nstable, and therefore, the growth disadvantage presumably comes from the overproduction of the large but incomplete and inactive galactosidase. The yield of somatosta~in has varied from 0.001 to 0.~3% of the total cellular protein (Table 1) probably as the result of the selection for cells in culture havin~ plasmids with a deleted lac region. The highest yields of somatostatin have been from preparations where growth was started from a single ampicillin reisistant, constitutive colony.
Even in these cases, 30% of the cells at harvest had deletions of the l~c region. Storage in the frozen state (lyophilization) and growth to harvest from a * trade mark ~2~ 3 single such colony is accordingly indicated for the system described. Yields may be increased by, e.g., resort to bacterial strains which overproduce lac repressor such that expression of precursor protein is essentially totally repressed prior to induction and harvest. Alternatively, as previously discussed, a tryptophan or other operator-promoter system which ordinarily is totally repressed may be employed.
In the crude extract resulting from cell disrup-tion in, e.g., an Eaton Press, the ~ -galactosidase -somatostatin precursor protein is insoluble and is found in the first low speed centrifugation pellet.
The activity can be solubilized in 7~% formic acid, 6M
guanidium hydrochloride, or 2~ sodium dodecyl sulfate.
Most preferably, however, the crude extract from the Eaton Press is extracted with 8M urea and the residue cleaved with cyanogen bromide. In initial experiements somatostatin activity derived from E. coli. strain RRl ~pSOM 11-3) has been enriched approximately 100-fold by alcohol extraction of the cleavage product and chromato-graphy on Sephadex G-50 in 50% acetic acid. When the product is again chromatographed on Sephadex G-50 and then subjected to high pressure liquid chromatography, substantially pure somatostatin may be obtained.
II. HUMAN INSULIN

The techniques previously described were next applied to the production of human insulin. Thus, the qenes for insulin B chain (104 base pairs) and for insulin A chain (77 base pairs) were designed from the amino acid sequence of the human polypeptides, each with single-stranded cohesive termini for the EcoRI
and BamHI restriction endonucleases and each designed for insertion separately into pBR322 plasmids. The synthetic fragments, deca- to pentadeca-nucleotides, were synthesized by the block phosphotriester method using trinucleotides as building blocks and ultimately 1 2Si~OA3 purified with high performance liquid chromatography (HPLC). ~he human insulin A and B chain synthetic genes were then cloned separately in plasmid pBR322.
The cloned synthetic genes were fused to an E. Coli ~ -galactosidase gene as before to provide efficient transcription, translation, and a stable precursor protein. Insulin peptides were cleaved from ~ -galac-tasidase precursor, detected by radioimmunoassy, and purified. Insulin radioimmunoassay activity was then generated by mixing the E. Coli products.

1. Design and Synthesis of Human Insulin Genes The genes constructed for human insulin are depicted in Figure 9. The genes or human insulin, B chain and A chain, were deslgned from the amino acid sequences of the human polypeptides. The 5' ends of each gene have single stranded cohesive termini for the Eco~I and BamHI restriction endonuclease~, for the correct insertion of each gene into plasmid pBR322. A ~indIII
endonuclease recognition site was incorporated into the middle of the B chain gene for the amino acid sequence Glu-Ala to allow amplification and verification of each half of the gene separately before the construction of the whole B chain gene. The B chain and the A chain genes were designed to be built from 29 different oligodeoxyribonucleotides, varying from decamer to pentadecamers. Each arrow indicates the fragment synthesized by the improved phosphotriester method, ~1 to H8 and Bl to B12 for the B chain gene and A1 to All for the A chain gene.

2. Chemical Synthesis of Oligodeoxyribonucleotides Materials and methods for synthesis of oligodeoxy-ribonucleotides were essentially those described in Itakura, R. et al (1975) J. Biol. Chem. 250, 4592 and Itakura, R. et al ~1975) J. Amer. Chem. S~c. 97, 7327 except for these modifications:

a) The fully protected mononucleotides, 5' 0-dimethoxytrityl-3'- ~-chlorophenyl- ~ -cyanoethyl phos-phates, were synthesized from the nucleoside derivatives using ~he monofunctional phosphorylating agent ~-chloro-phenyl- p -cyanoethyl phosphorochloridate (1.5 molar equivalent) in acetonitrile in the presence of l-methyl imidazole Van Boom, J.~. et al (1975) Tetrahedron 31, 2953. The products were isolated in large scale (1~0 to 3009) by preparative liquid chromatography (Prep 500 0 LC, Waters Associates).
b) By using the solvent extraction method [Hirose, T. et al (1978) Tetrahedron Letters, 2449] 32 bifunctional trimers were synthesized (see Table IV) in 5 to 10 mmole scale, and 13 trimers, 3 tetramers, and 4 dimers as the 3' terminus blocks, in 1 mmole scale.
The homogeneity of the ~ully protected trimers was checked by thin layer chromatography on silica gel in two m~thanol/chloroform solvent systems: solvent a, 5%
v/v and solvent b, 10% v/v (See Table IV). Starting from this library of compo~nds, 29 oligodeoxyribonucleo-tides of defined sequence were synthesized, 18 for the B chain and 11 for the A chain gene.
The basic units used to construct polynucleotides were two types of trim~r block, i.e. the bifunctional trimer blocks of Table IV and corresponding 3'-terminus trimers protected by an anisoyl group at 3'-hydroxy.
The bifunctional trimer was hydrolyzed to the correspon-ding 3'-phosphodiester component with a mixture of pyridine-triethylamine-water (3:1:1 v/v) and also to the corresponding 5'-hydroxyl component with 2~ benzene-sulfonic acid. The 3'-terminus block previously referred to was treated with 2% benzenesulfonic acid to give the corresponding 5'-hydroxyl. The coupling reaction of an excess of the 3'-phosphodiester trimer (1.5 molar equivalent) with the 5'-hydroxyl component, however obtained~ (1 molar equivalent) in the presence of 2,4,6-triisopropylbenzenesulfonyl tetrazolide (TPSTe, 3 to 4 equivalents) w~nt almost to completion in 3 hours. To remove the excess of the 3'-phospho-TABLE IV
SYNTHESIS OF TRI~ER BUILDING BLOCRS

No Compound* Yield** Rf Purity*** In Figure 9, 5 ~ ) a. b. ~%) Present In-1. AAG 47 0.15 0.40 93 B5,B6 2. AAT 49 0.25 0.52 95 ~l,Al,A6 3 AAC 52 0.28 0.55 93 H5,86,A2,A8 4 ACT 43 0.27 0.53 91 B4,B5,A6 5. ACC 56 0.33 0.60 96 B7 6. ACG 39 0.18 0.45 90 H5,B7 107. A~G 45 0.10 0.26 89 ~6,~7,B9 8. AGT 33 0.14 0.40 96 B9,A2tAll

9. ACC 50 0.19 0.48 92 ~8,Bl,A5,A10

10. AGA 48 0.24 0.50 91 A9,

11. TTC 44 0.26 0.52 95 B4,B7,A3

12. TTC 49 0.11 0.31 94 H3,~5,A2,A3tA5

13 TCT 58 0.24 0.49 96 A4 1514 TCA 45 0.28 0.53 92 ~ 2,H4,A1 15. TCG 39 0.12 0.34 91 A2 16 TGG 32 0.10 0.28 87 H3,Al,A10 17 TGC 51 0.18 0.47 93 H6,B2,A4,A7,A8 18. TGA 46 0.12 0.37 94 H7 19. TAC 61 0.22 0.50 90 B4,All 20. TAA 55 0.17 0.44 95 B5,A10 21. CCT 53 0 30 0.55 97 H3,H4,B10 2022 CAC 47 0.25 0.51 92 A3 23 CAA 58 0.25 0.51 93 H2,H6,H8,A7 24. CTT 41 0.28 0.54 92 B2,B9,A4 CGA 40 0.27 0.52 93 A7 26 CGT 75 0.25 0.50 89 H2,~4,B3,Bl 27. GGT 35 0.09 0.26 90 B3 28. GTT 46 0.18 0.45 93 B2 2529. GTA 38 0.25 0.50 95 B6,B8,A6 30. GAA 39 0.15 0.39 88 H7,B3lB8,A5 31 GAT 52 0.22 0.49 89 B10,A9 32 GCA 42 0.14 0.39 93 A9 * Fully protected trideoxynucleotides; 5-0-~imethoxytrityl-3'~ p-Chlorophenyl-~ -cyanoethyl 30 phosphate.
** Yield was the overall yield calculated from the 5'-hydroxylmonomers.
*** Based on ~PLC analysis.

~L2~ 3 -3~-diester block reactant the reaction mixture was passed through a short silica gel column set up on a sintered glass filter. The column was washed, first with CHC13 to elute some side products and the coupling reagent, and then with CHC13:MeOH (95:5 v/v) in which almost all of the fully protected oligomer was eluted.
Under these conditions, the charged 3'-phosphodiester block reactant remained in the column. Similarly, block couplings were repeated until th desired length was constructed.
~ igh performance liquid chromatography (~PLC) was used extensively during oligonucleotide synthesis for a) analysis of each trimer and tetramer block~ b) analysis of the intermediate fragments (hexamers~
15 nonamers, and ~ecamers) J C) analysis of the last coupling reaction, and d) purification of the final product~. The HPLC was performed by using a Spectra-Physics 3500B liquid chromatograph. After removal of all protecting groups by conc. NH40H at 50C (6 h) 20 and 80% AcOH at room temperature (15 min), the compounds were analyzed on a Permaphase AAX (DuPont) ~Van Boom, J. et al (1977) J. Chromatography 131, 169.] column (1 mX 2 mm), using a linear gradient of solvent B (0.05M
RH2P04 -l.OM KCl,pH 4.5) in solvent A (O.OlM
RH2P04, pH 4.5). The gradient was formed starting with buffer A and applying 3~ of buffer B per minute.
The elution was performed at 60C, with a flow rate of 2 ml per minute. The purification of the 29 final oligonucleotides also was performed on Permaphase AAX, 30 under the same conditions reported above. The desired peak was pooled, desalted by dialysis, and lyophilized.
After labeling the 5' termini with (~-32P)ATP using T4 polynucleotide kinase, the homogeneity of each oligonucleotide was checked by electrophoresis on a 20%
35 polyacrylamide gei.

* Trademark ~l2S~0~3 -3g-3. Assembly_and Cloninq of B Chain Gene and the A Chain_ Gene The gene for the B chain of insulin was designed to have an EcoRI restriction site on the left end, a HindIII site in the middle and BamHI site at the right end. This was done so that both halves, the left EcoRI-HindIII half (BH) and the right indIII-BamHI
half (BB), could be separ~tely cloned in the convenient cloning vehicle pBR322 and after their sequences had been verified, joined to give the complete B gene (Figure 10). The BB half was assembled by ligation from 10 oligodeoxyribonucleotides, labeled Bl to B10 in Figure 9, made by phosphotriester chemical syn~hesis.
Bl and B10 were not phosphorylated, thereby eliminating unwanted polymerization of these fragments through their cohesive ends (HindIII and BamHI). After puri-fication by preparative acrylamide gel electrophoresis and elution of the largest DNA band, the BB fragment was inserted into plasmid pBR322 which had been cleaved with HindIII and BamHI. About 50~ of the ampicillin resistant colonies derived from the DNA were sensitive to tetracycline, indicating that a nonplasmid HindIII-BamHI fragment had been inserted. The small HindIII-BamHI fragments from four of these colonies (pBB101 to pBB104) were sequenced and found to be correct as designed.
The BH fragment was prepared in a similar manner and inserted into pBR322 which ~ad been cleaved with EcoRI and HindIII restriction endonucleases. Plasmids from three ampicillin resistant, tetracycline sensitive transformants (pBHl to pBH3) were analyzed. The small EcoRI-HindIII fragments were found to have the expected nucleotide sequence.
The A chain gene was assembled in three parts.
The left four, middle four, and right four oligonucleo-tides (see Figure 9) were ligated separately, then mixed and ligated (oligonucleotides Al and A12 were unphosphorylated). The assembled A chain gene was .

s~

phosphorylated, purified by gel electxophoresis, and cloned in pBR322 at the EcoRI-BamHI sites. The EcoRI-BamHI fragments from two ampicillin resistant, tetra-cycline sensitive clones (pA10, pAll) contained the S desired A gene sequence.

4. Construction of Plasmids for Ex ression of A and B
Insulin Genes Figure 10 illustrates the construction of the lac-insulin B plasmid (pIBl). Plasmids pBHl and pBB101 were digested with EcoRI and ~indIII endonucleases.
The small BH fragment of pBHl and the large fragment of pBB101 (containing the BB fragment and most of pBR322) were purified by gel electrophoresis, mixed, and ligated in the presence of EcoRI-cleaved ~ plac5. The megadal-ton EcoRI fragment of ~plac5 contains the lac control region and the majority of the ~ -galacto~idase structural gene. The configuration of the restriction sites ensures correct joining of BH to BB. The lac EcoRI fragment can insert in two orientations; thus, only half of the clones obtained after transformation should have the desired orientation. The orientation of ten ampicillin resistant, ~ -galactosidase consti-tutive clones were checked by restriction analysis.
Five of these colonies contained the entire B gene sequence and the correct reading frame from the ~ -galactosidase gene into the B chain gene. One, pIBl, was chosen for subsequent experiments.
In a similar experiment, the 4.4 megadalton lac fragment from ~plac5 was introduced into the pAll plasmid at the EcoRI site to give pIAl. pIAl is identi-cal to pIBl except that the A gene fragment is substi-tuted for the B gene fragment. DNA sequence analysis demonstrated that the correct A and B chain gene sequences were retained in pIAl and plBl respectivelyO

5. Expression The strains which contain the insulin genes correctly attached to ~ -galactosidase both produce large quantities of a protein the size of ~ -galacto-sidase. Approximately 20~ of the total cellularprotein was this ~ -galactosidase-insulin A or B chain hybrid. The hybrid proteins are insoluble and were found in the first low speed pellet where they consti-tute about 50% of the protein.
To detect the expression of the insulin A and B
chains, we used a radioimmunoassay (RIA) based on the reconstitution of complete insulin from the separate chains. The insulin reconstitution procedure of Ratsoyannis et al ~1967) Biochemistry 6, 2642 -2655, adapted to a 27-microliter assay volume, provides a very suitable assay. Easily detectable insulin activity is obtained after mixing and reconstituting S-Sulfonated derivatives of the insulin chains. The separate S-sul-fonated chains of insulin do not react significantly, after reduction and oxidation, with the anti-insulin antibody used.
To use the reconstitution assay we partially purified the ~-galactosidase-A or B chain hybrid protein, cleaved with cyanogen bromide, and formed S-sulfonated derivatives.
The evidence that we have obtained correct expres-sion from chemically synthesized genes for human insulin can be summarized as follows: a) Radioimmune activity has been detected for both chains. b) The DNA
sequences obtained after cloning and plasmid construction have been directly verified to be correct as designed.
Since radioimmune activity is obtained, translation must be in phase. Therefore, the genetic code dictates that peptides with tbe sequences of human insulin are being produced. c) The E. coli products, after cyanogen bromide cleavage, behave as insulin chains in three different chromatographic systems which separate on different principles (gel filtration~ ion exchange, and reversed phase H~LC). d) The E coli produced A chain ~L~S~

has been pur if ied on a small scale by HPLC and has the correct amino acid composition.

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Claims (29)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A recombinant microbial cloning vehicle comprising a regulon, a DNA sequence for a polypeptide including a first amino acid sequence and a second amino acid sequence, and one or more termination codon(s), wherein codons coding for the second amino acid sequence are interposed between said regulon and termination codon(s) without altering the reading frame of said DNA sequence such that a polypeptide comprising both the first and second amino acid sequences results from expression, there being a selective cleavage site adjacent the first amino acid sequence.

2. A cloning vehicle as claimed in claim 1 , wherein the selective cleavage site is in the second amino acid sequence.

3. A vehicle according to claim 1 wherein the first amino acid sequence contains no similar selective cleavage site.

4. A vehicle according to claim 3, which is a bacterial plasmid and wherein the recited elements are sequenced as follows: (regulon) - (codons for second amino acid sequence) -(codons for first amino acid sequence) - (termination codon(s)).

5. A vehicle according to claim 4 , wherein the cleavage site is methionine.

6. A vehicle according to claim 5 , wherein the first amino acid sequence is somatostatin.

7. A vehicle according to either claims 1 or 2 , wherein the first amino acid sequence codes for the A chain of insulin or the B chain of insulin.

8. Plasmid pSOM1.

9. Plasmid pSOM11,

10. Plasmid pSOM11-3.

11. Plasmid pIA-1.

12. Plasmid pIB-1.

13. A microorganism transformed with a cloning vehicle acccrding to claim 1 .

14. A microorganism transformed with a plasmid accor-ding to claim 8.

15. A mirroorganism transformed with a plasmid accor-ding to either of claims 11 or 12.

16. A microbial culture comprising a transformed microorganism according to either claim 13.

17. A microbial culture comprising a transformed mi-croorganism according to claim 14 .

18. A microbial culture cloned from one or more host microorganisms each comprising a cloning vehicle according to Claim 1 , the members of said culture being capable of expressing said polypeptide.

19. A microbial culture cloned from one or more host microorganisms each comprising a plasmid according to any one of claims 10 to 12, the members of said culture being capable of expressing said polypeptide.

20. A microbial culture as claimed in claim 18 , wherein the host microorganism is a bacterium.

21. A transformant bacterial culture cloned from one or more bacteria each comprising cloning vehicles as claimed in any one of claims 1 or 2, the members of said culture being capable of expressing the polypeptide.

22. A transformant bacterial culture cloned from one or more bacteria each comprising plasmids as claimed in any one of claims 10 to 12, the members of said culture being capable of expressing the polypeptide.

23. A bacterial culture comprising E. Coli. RR1 (pSOM11-3).

24. A bacterial culture comprising E. Coli. 294 (pIA-1).

25. A bacterial culture comprising E. Coli. 31210 (pIB-1).

26. A vehicle according to claim 2 wherein the first amino acid sequence contains no similar selective cleavage site.

27. A microorganism transformed with a cloning vehicle according to claim 2.

28. A microorganism transformed with a plasmid according to claim 9 or 10.

29. A microbial culture cloned from one or more host microorganisms each comprising a cloning vehicle according to claim 2, the members of said culture being capable of expressing said polypeptide.