IE50833B1

IE50833B1 - Recombinant dna techniques

Info

Publication number: IE50833B1
Application number: IE71/81A
Authority: IE
Original assignee: Searle & Co
Priority date: 1980-01-30
Filing date: 1981-01-15
Publication date: 1986-07-23
Also published as: BE887290A; CA1189466A; JPS579795A; SE451595B; IL61952A0; SE8100238L; NL8100437A; FR2479259A1; IE810071L; FR2479259B1; IT8147639A0; CH662821A5; IL61952A; DE3102721C2; DK17881A; AU6650781A; AU545908B2; ES498948A0; DE3102721A1; ES8203965A1

Abstract

The invention relates to a synthetic gene which contains in the coding strand codons for the amino acids of a required peptide in tandem repeats. The invention further comprises a process of a synthetic gene of this type, which is characterised by self-attachment and ligation and, where appropriate, repair of a plurality of suitable oligonucleotides, where the attachment is achieved with the formation of the repetitive character by self-complementary, overlapping ends at the termini of the group of oligonucleotides which, after the attachment, form the repetitive unit of the gene. A synthetic gene of this type can be used to prepare recombinant plasmid vectors, to transform cells and thus to express peptides. The invention is applicable, inter alia, to the preparation of (asparagyl-phenylalanine)n and, from this, the sweetener aspartame, that is to say the methyl ester of asparagyl-phenylalanine.

Description

This invention relates to recombinant DNA techniques.

More particularly, in its broadest aspect, the present invention relates to a process for the preparation of oligopeptides or polypeptides by recombinant DNA techniques using a synthetic gene coding for a repeating polymer of the desired oligopeptide or polypeptide.

The present invention also relates to such a synthetic gene and to the preparation thereof.

The synthesis of peptides by chemical means is a laborious process, which becomes progressively less efficient with increasing length of the peptide. On the other hand, the preparation of peptides by microorganisms in a fermentation process does not vary in efficiency in relation to the length of the peptide product. However, in the organisms used in recombinant DNA processes, short peptides are metabolically unstable. It is known, for example, that the peptide somatostatin, which is composed of 14 amino acids, is not stable when produced directly in E. coli. Stability is achieved in that case by linking the peptide to a normal E. coli. protein and subsequently cleaving this fusion product. (Itakura, K., et al, Science, 198, 1056 - 1063, (1977)).

In the present process, stability and increased yield are achieved by the use of a synthetic gene «0833 -3containing multiple repeats of the sequence coding for a desired small peptide. The product is thus a large molecule which is metabolically more stable in E. coli than the small peptide. Subsequent cleavage of the large molecule gives the smaller peptide contained within it and in higher molar yield than would be obtained if the small peptide had been prepared directly using a monomeric gene even if the small peptide were metabolically stable.

In one embodiment, the present invention relates to a synthetic gene which comprises, in the coding strand, codons for the amino acids of a desired peptide in tandem repeat.

By the term tandem repeat in this context is meant the repeat of the sequence of codons in a linear progression along the DNA molecule with the same polarity and without interruption.

The complementary or non-coding strand of the synthetic gene contains a sequence specified by the sequence of the coding strand according to the accepted rules of nucleic acid base pairing.

In another embodiment, the present invention relates to a process for the preparation of such a synthetic gene which comprises the self-assembly and ligation and optionally repair of a plurality of appropriate oligonucleotides, the assembly to give the repeating character being achieved by self-complementary -4overlapping ends at the termini of the group of oligonucleotides which, when assembled, comprises the repeating unit of the gene.

In a further embodiment, the present invention relates to a plasmid vector which comprises inserted therein at an insertion site adjacent to a bacterial promoter and downstream from a prokaryotic ribosome binding site such a synthetic gene such that the gene is under bacterial promoter control.

In yet another embodiment, the present invention relates to a cell which has been transformed by having inserted therein such a plasmid vector.

In a further embodiment, the present invention relates to a process for the expression of a peptide which comprises culturing such a cell.

As indicated above, the strands of the synthetic gene are prepared from a series of short oligonucleotides which themselves are prepared by joining together the required nucleotides by known methods in an order specified by the codons of the amino acids of the desired peptide and the rules set out in more detail below.

The oligonucleotides of the coding strand and the complementary strand of the gene form an overlapping set corresponding to the following general structure:50833 The structure shown in this illustration is composed of six oligonucleotides, three in each strand. The structure may, however, be assembled from any number of pairs of oligonucleotides from one pair upwards, provided:(1) that the paired regions designated a^ 1 2 1 2 and a , b and b , c and c — contain nucleotide base sequences which are complementary within the pair, but not to the sequences of any other pair; 12 (2) that each of these regions a , a , b , b , 1 2 c , c ... contain at least four nucleotide bases; and (3) that the sum of the nucleotide bases in each strand, which when assembled eventually comprises the repeat length of the polymer, is divisible by three.

When a set of oligonucleotides of this character are mixed, the complementary regions of the oligonucleotide pairs hybridize to form structures of high molecular weight in which the sequences of the oligonucleotides (a^-f1 in the above illustration) are repeated in tandem from a few -6times to many times, in different molecules. In the subsequent process, it is desirable to maximise the number of polymers which have in the sequence a^ at the 5' termini thereof. This is achieved by mixing 11 11 11 22 the oligonucleotides a b,c d,e f,b c and 2 2 d e of the above illustration in equimolar amounts 2 2 and subsequently adding the oligonucleotide f a in a less than equimolar amount. Since the complements of a1 and f1 are present in sub-molar amounts, most polymers will terminate in a^ and f1. In the general case, the 5' terminal oligonucleotide of the comple0 mentary set is added to the polymerization mixture in an amount which is less than equimolar with respect to the other oligonucleotides, all of which are present in equimolar amounts.

The nicks in the chains, where adjacent oligonucleotides in the chain juxtapose, may be joined by the use of the enzyme DNA ligase (EC 6.5.1.1), a known procedure.

The thus-prepared molecules will have single stranded ends. These may be converted to the so-called blunt end form by use of the enzyme DNA polymerase I (EC 2.7.7.7) by a known procedure.

The resultant molecules may be cloned in competent microbiol cells, in particular E coll, using a desired vector. In particular, a plasmid vector selected from -7the so-called pWT series, see, for example, Tacon, et a 1, Molec. Gen. Genet., 177, 427, (1980), may be used, selection of the vector being made such that the synthetic gene, when inserted in the correct orientation, is read in the desired translation reading frame.

Figure 1 of the accompanying drawings illustrates a scheme for the production of a recombinant plasmid (pWT 121 (asp-phe)n) which contains the repeating gene for (asp-phe)n in the correct DNA phasing for expression of an (asp-phe)n protein. The plasmid DNA, shown at the top of the diagram, is cut with the restriction enzyme Hind III and then repaired with E. coli DNA polymerase I to produce a blunt-ended molecule. The polymer coding for (asp-phe)n, shown at the bottom of the diagram, has its initially-protruding 5* -end repaired similarly to produce another blunt ended molecule. The joining of these two blunt ended species with T4 DNA ligase produces the recombinant DNA, shown in the centre of the diagram, having the correct DNA phasing for expression of (asp-phe)n· The cloning of this gene, the selection of bacterial colonies, the detection of the polypeptide product of the expression of the gene and the extraction and purification of the polypeptide may all be achieved by known procedures.

This polypeptide may then be cleaved either enzymically using specific enzymes, or chemically, for -8 example by the use of cyanogen bromide to cleave polypeptide chains at methionine residues.

As will be appreciated by those skilled in the art, the present invention finds numerous applications.

Inter alia, the present invention may be applied to the peptide hormones oxytocin, vasopressin and somatostatin, the enkephalins (opiate pentapeptides) and the appetitecontrolling peptides.

For purposes of illustration one particular 10 embodiment of the present invention will now be described in more detail with reference to the sweetener Aspartame Aspartame is a low calorie artificial sweetener which may be described chemically as aspartylphenylalanine -9methyl ester. Aspartame is currently produced by a multi-stage chemical synthesis.

In view of its use as a sweetener, the total consumption of Aspartame is potentially very large and there is, therefore, considerable interest in developing preparations whereby the material may be obtained conveniently and on a large scale.

It has been found that Aspartame may be prepared by a potentially large scale process based upon recombinant DNA techniques whereby a synthetic gene coding for (aspartyl-phenylalanine) is inserted into a cloning vector having a controllable bacterial promoter upstream of and substantially adjacent to the insertion site and the vector may be used to transform hacterial cells from which the expressed polypeptide may be obtained and this may then be cleaved to obtain aspartylphenylalanine and hence Aspartame.

Accordingly, in a further embodiment, the present invention relates to a synthetic gene for the expression of the repeating polypeptide (aspartyl-phenylalanine) which comprises a double-stranded DNA molecule comprising in the coding strand at least two nucleotide trimers coding for the expression of aspartic acid (Asp) and, alternating therewith, at least two nucleotide trimers coding for the expression of phenylalanine (Phe) and in the other strand repeating and alternating nucleotide sequences complementary to the (Phe) and (Asp) coding sequences. -10The coding strand of the synthetic gene, i.e. that coding for the repeating polypeptide (Asp-Phe)n, may therefore by represented as coding: ' - (Asp - Phe - Asp - Phe)n - 3' wherein n represents an integer, e.g. of up to several hundred.

There are two possible codons for Phe, viz (T-T-T) and (T-T-C), and two possible codons for Asp, viz (G-A-T) and (G-A-C), and these may be used in any permutation with corresponding complementary sequences in the other strand. For example, the coding strand may be a nucleotide sequence as follows: Phe Asp Phe Asp Phe Asp 5ι _T_T_T_C_G_A_C_T_T_C_G_A_T_T_T_C_G_A_C_T_T_ 3ι with a complementary strand having the sequence 3' -A-A-A-G-C-T-G-A-A-G-C-T-A-A-A-G-C-T-G-A-A- 5' In this case, (T-T-C) Is used as the sequence coding for phenylalanine and both of the codons (G-A-C) and (G-A-T) alternate for aspartic acid.

The other possible permutations and combinations of nucleotide sequence for the coding and complementary strands will be clear to those skilled in the art.

The synthetic Aspartame gene according to the present invention may be prepared by the polymer25 isation of a double stranded fragment of DNA consisting of two dodecanucleotide strands, one having the sequence for expression of (Asp-Phe)2, the coding strand and the -11other being complementary to it, the complementary strand.

Accordingly, in a further embodiment, the present invention relates to such a process which comprises providing a first dodecanucleotide sequence coding for aspartyl-phenylalanine by linking together appropriate nucleotide monomers, providing a second dodecanucleotide sequence complementary to the first sequence, phosphorylating both dodecanucleotides using polynucleotide kinase and mixing the first and second sequences together to form a double-stranded DNA structure.

The dodecanucleotide strands may each be synthesised from the respective monomers which are protected by blocking groups in known manner and linked by conventional phosphotriester methodology, (see, for example, Hsiung, et al, Nucleic Acids Research, 6, 1371 1385, (1979)), the dodecamers so obtained are deblocked and subsequently purified. Following purification and phosphorylation with polynucleotide kinase (E.C. 2. 7.1.78), the coding and complementary strands are mixed together with the result that double stranded structures are formed by hydrogen bonding. It has been found that, by using the dodecamer coding for (Asp-Phe)2 for the coding strand and a corresponding dodecanucleotide complementary strand, very stable double-stranded structures are obtained resulting from the six base overlap of the strands, which, for the example -12previously given, is represented as follows: ' pT-T-T-C-G-A-C-T-T-C-G-A 3' 3' G-A-A-G-C-T-A-A-A-G-C-Tp 5' Linear polymerisation of the above double-stranded structures is achieved by incubation with T^-DNA ligase (EC 6.5.1.1) to yield long polymers of random length. After separation of unligated material, it is found that a range of polymers is obtained containing from 2 to 500 and more repeats of the basic units.

The present invention also relates to a process for the expression of (aspartyl-phenylalanine) which comprises inserting such a synthetic gene into an insertion site of a plasmid vector reading in the correct phase for the inserted gene, the insertion site being adjacent to a bacterial promoter and downstream from a prokaryotic ribosome binding site, transforming competent microbial cells using the ’ resulting plasmid vector, culturing the transformed cells and harvesting the expressed peptide. Moreover, the polymeric product is considered novel.

The present invention further relates to a process for the preparation of Aspartame (the methyl ester of aspartyl-phenylalanine) which comprises inserting the above-mentioned synthetic gene into a plasmid cloning vector, e.g. pWT 121, designed to read in the correct phase for expression of the inserted gene and having a bacterial promoter upstream of and -13adjacent to the insertion site. The plasmid vector may be used to transform E. coli HB 101 cells, the expressed (Asp-Phe)n harvested and subjected to enzymatic cleavage using an enzyme specific for amino acids with aromatic side chains, such as subtilisin (E.C. 3.4.4.16), chymotrypsin (E.C. 3.4.4.5) or proteinase K (E.C. 3.4.21.14), to obtain aspartylphenylalanine which is methylated to produce Aspartame. The enzyme used may be in a soluble form or, preferably, immobilised on a solid support. Methylation may either be carried out by conventional chemical means or by an exchange reaction with the enzyme in the presence of methanol or by direct enzymatic methanolysis of the polymer.

It will be appreciated that, given the triplet nature of the genetic code, the gene may be read in any one of three phases and it is therefore essential for a gene to use as the plasmid vector one providing for translation in the correct reading frame.

It has been found that a preferred plasmid vector for use in the present process is one selected from the so-called pWT series mentioned above.

Basically, the pWT series plasmids comprise a Hin d III (E.C. 3.1.23.21) restriction site adjacent to a cloned E. coli tryptophan promoter. By Hin d III restriction and the insertion of Hin d III linkers it is possible in the pWT series to construct plasmids able -14to translate in all three reading frames these being pWT 111, pWT 121 and pWT 131 as disclosed in the abovementioned reference.

Transcription and translation of inserted DNA 5 in the pWT series plasmids is under direct and strong tryptophan control; thus, in the presence of tryptophan, the operon is repressed, while, in the absence of tryptophan, it is de-repressed.

Plasmids of the pWT series also possess, in the region immediately following the Hin d III site, the gene for tetracycline resistance so that, after insertion of the DNA, transcription and translation may easily be confirmed since, when it has occurred, tetracycline resistance is maintained. in a preferred embodiment of the present process, therefore, the synthetic gene is inserted into the appropriate plasmid of the pWT series, that is the plasmid designated pWT 121. This plasmid is illustrated diagrammatically in Figure 2 of the accompanying drawings and has the following characteristics: A molecular length of 4837 bp; a Hpa I (E.C. 3.1.23.23) sate; a Hin d III site 206 bp from the Hpa I site; a Bam HI (E.C. 3.1.23.6) site 353 bp from the Hin d III site; a Sal I (E.C. 3.1.2337) site 275 bp from the Bam HI site; a Pst I (E.C. 3.1.23.31) site 2958 bp from the Sal· I site and 1045 bp from the Hpa I site; the gene for tetracycline resistance extending from the region -15of the Hpa I site to beyond the Sal I site; the gene for ampicillin resistance in the region of the Pstl I site and the cloned portion of the trp operon comprising the region between the promoter and the first portion of the E gene between the Hpa I and the Hin d III sites.

The synthetic Aspartame gene according to the present invention was cloned into pWT 121 as follows:The plasmid pWT 121 was restricted with the enzyme Hin d III to yield a linear molecule which was then treated with DNA polymerase and all four deoxyribonucleoside triphosphates to produce completely base paired 'blunt ends'. The blunt-ended molecule was then treated with bacterial alkaline phosphatase (E.C.3.1.3.1) to remove the 5' phosphates. The plasmid is now prepared for insertion of the synthetic gene which is first treated as follows :The synthetic gene is treated with E. coli DNA polymerase and all four deoxyribonucleoside triphosphates to produce blunt ends and the repaired genes separated from enzyme and small molecules.

Blunt ended vector DNA and blunt ended gene material are mixed in a from 1:1 to 1:2 ratio and ligated together with high concentrations of T4~DNA ligase to produce the series of plasmids pWT 121/(asp25 phe)n. The sizes of the cloned inserts/, determined by restriction enzyme digestion, were found to range from 60 to 900 bp (i.e. from 5 to 75 repeats of the dedecanucleotide unit). -16The pWT 121/(asp-phe) plasmids prepared as described above are used to transform E. coli cells in known manner, for example by exposure of cells treated with calcium chloride to the pWT 121/(asp-phe) plasmid. Transformed cells cultured in known manner in medium containing no tryptophan and preferably containing the inducer 6-indolearcylic acid, expressed a protein consisting essentially of (asp-phe)n which, after enzymatic cleavage and methylation, produced the desired aspartyl-phenylalanine methyl ester. Aspartame.

As mentioned above, the product Aspartame is conventionally obtained by a multi-stage chemical synthesis. An intermediate used in this conventional synthesis is L-phenylalanine. The present invention also relates to the preparation of L-phenylalanine useful in this conventional synthesis. When produced by chemical synthetic means, phenylalanine is obtained as a racemate which needs to be resolved before the L-isomer may be obtained and this requires an additional and expensive stage in the process. When obtained by an embodiment of the present invention, the phenylalanine is produced directly as the L-isomer.

Accordingly, the present invention further relates to a process for the preparation of L-phenylalanine which comprises treating the (asp-phe) peptide, isolated from a culture of E. coli cells carrying plasmids incorporating the synthetic Aspartame gene, with an -17acidic or enzymatic hydrolysing agent and separating from the hydrolysate the desired L-phenylalanine.

This process, of course, also gives rise to L-aspartic acid and the present invention also relates to such a process.

Preferred hydrolysing agents include hydrochloric acid or carboxypeptidase (E.C. 3.4.12.x), aminopeptidase (E.C. 3.4.11.x) or non specific endopeptidases.

Separation of the hydrolysis products may be achieved by known methods.

The following Examples illustrate the present invention: EXAMPLE 1 (asp-phe) Synthesis of dodecanucleotides The two dodecanucleotides, TpCpGpApApApTpCpGpApApG and TpTpTpCpGpApCpTpTpCpGpA, were synthesised by conventional triester methodology as described, for example, by Hsiung, loc cit, and in accordance with the reaction scheme illustrated in accompanying Figure 3 wherein N represents Gisol3U, T, Cbz or Abz. The fully protected dodecanucleotides were deblocked with 2% w/v benzene sulphonic acid, 0.1 M tetraethyl ammonium fluoride in THF/pyridine/water (8:1:1 by volume), followed by treatment with ammonia. The de-blocked dodecanucleotides were purified by HPLC on Partisil (Registered Trade Mark) SAX, (micro-particulate silica which has been derivatised with quaternary ammonium groups (Whatmann)), -18Polymerisation of dodecanucleotides Each synthetic dodecanucleotide (2 ug) was phosphorylated in a 10 pi reaction containing from 10 to 50 pCi of γ_32Ρ-ΑΤΡ, 50 mM Tris-Cl pH 7.8, 5 mM magnesium chloride, 0.25 mM unlabelled ATP, 10 mM mercaptoethanol and 3 units (1 unit is the amount that Ί 32 causes the transfer of 1 nanomole of P-phosphate from f 32 γ P)-ATP to the 5' hydroxy terminus of a polynucleotide in 30 minutes at 37°C under standard assay conditions, see, for example, Richardson, Nucleic Acids Research, 2, 815) of T^ polynucleotide kinase (E.C. 2.7.1.78). After 60 minutes at 37°C the two dodecanucleotides were mixed, unlabelled ATP was added to bring the ATP up to 1 mM together with 0.1 units (1 unit is the amount that will convert 100 nanomoles of d(A-T)10Q0t0 an exonuclease Hires is tant form in 30 minutes at 30°C under standard assay conditions, see, for example, Modrich and Lehman, J.

Biol. Chem., 245, 3626 (1970)) of T4 DNA ligase (E.C. 6.5. 1.1) and the reaction mixture incubated for 24 hours at °C. ATP and unligated nucleotide monomers were removed by passage down a Sephadex (Registered Trade Mark) G-50 (superfine, particulate cross-linked modified dextran polymer (Pharmacia)) column (20 cm X 0.8 cm) in 50 mM sodium chloride, 10 mM Tris-Cl pH 7.5, 0.2% w/v sodium dodecylsulphate (SDS). The double-stranded polymers were concentrated by ethanol precipitation. The product, after separation, was found to be a mixture of polymers of -19random length containing from 2 to more than 500 repeats of the basic units.

Cloning of the synthetic gene (a) Blunt end repair of the synthetic gene 2 jig of polymer prepared as described above was incubated in a 40 pi mixture with 50 mM Tris-Cl pH 7.8, 5 mM magnesium chloride, 1 mM mercaptoethanol, 0.125 mM of each of the four deoxynucleotide triphosphates and 2 units (1 unit is the amount that causes the incorporation of 10 nanomoles of nucleotide into an acid precipitable form in 30 minutes at 37°c under standard assay conditions using poly d (A-T) as template, see, for example, Richardson, et al, J. Biol. Chem., 239, 222, (1964)) of coll DNA polymerase X (E.C. 2.7. 7.7). The mixture was incubated for 20 minutes at 10°C and the mixture used without further purification. (b) Preparation of the cloning vector pWT 121 fig of pWT 121 DNA was digested with a two-fold excess of Hin d III (E.C. 3.1.23.21). The mixture was extracted twice with an equal volume of phenol and precipitated with ethanol.

Repair with E. coli DNA polymerase to generate blunt ends was carried out as described in (a) above.

The blunt ended DNA was then treated with bacterial alkaline phosphatase (E.C. 3.1.3.1) to remove terminal phosphates and to prevent the DNA recircularising during subsequent ligation. The DNA was incubated for 30 -20minutes at 37°C in the presence of a two-fold excess of enzyme in 10 mM Tris-Ci pH 7.5, 0.1% SDS. The mixture was thereafter exhaustively extracted with phenol, washed several times with chloroform and then precipitated with ethanol. (c) Blunt end ligation Blunt ended dodecanucleotide polymer from (a) above and blunt ended pWT 121 DNA from (b) above were mixed in a 1:1 proportion, by weight, and ligated at °C with 0.2 units of T^ DNA ligase in a 20 pi mixture containing 50 mM Tris-Cl pH 7.8, 5 mM magnesium chloride, mM ATP and 10 mM mercaptoethanol. After 24 hours the pWT 121/(asp-phe)nrecombinant plasmids were ready for use to transform E. coli cells.

Transformation and gene expression E. coli K12 HB 101 cells (genotype gal , lac , ara , pro , arg , strr, rec A , r^ , Mj, ; Boyer, H.W. and Roullard - Dussoix, D., J. Mol. Biol., 41, 459-472) were transformed by the procedure of Katz et al (1973) J. Bacteriol, 114, 577-591, and plated on L-agar plates supplemented with ampicillin (100 pg/ml).

Recombinant clones were purified by streaking to obtain single colonies. Clones thus isolated were examined by colony hybridisation on nitro-cellulose filters (Grunstein and Hogness (1975) Proc. Nat. AcadSci. U.S.A. 72, 3961-3965) using a kinase-labelled -21synthetic dodecanucleotide as a hybridisation probe.

Single colonies positive by colony hybridisation were grown up to an of 0.6 in 25 ml of M9 medium (Miller, (1972), Experiments in Molecular Genetics, Cold Spring Harbour Laboratory, New York, page 433) supplemented with ampicillin (100 pg/ml) and tryptophan (40 pg/ml).

A 1 ml sample was taken from this repressed culture and 14 labelled for 10 minutes with 5 pCi C-amino acids before being chased for 10 minutes with 200 pi of 20% w/v casamino acids (obtainable frcm Difco). The cells were centrifuged (pelleted) (10,000 x g for 10 minutes), washed several times with phosphate-buffered saline containing 100 pg/ml gelatin to remove excess label and the final pellet lysed in 50 pi of a buffer (FSB) containing 10% v/v glycerol, 0.01% w/v bromophenol blue, 5% v/v β-mercaptoethanol, 3% w/v SDS and 65 mM Tris-Cl pH 8 by heating to 90°C for 2 minutes. The remainder of the 25 ml initial culture was pelleted (10,000 x g for 10 minutes) and resuspended in an equal volume of M9 medium supplemented with ampicillin (100 pg/ml) and β-indoleacrylic acid (5 pg/ml). After various times of induction 1 ml samples were withdrawn and labelled as described above. From 5 to 10 pi aliquots of the final lysates were separated on acrylamide gels (12.5% polyacrylamide + 0.1% SDS, see, for example, Laemmli, Nature, 227, 680-685, (1970)).

The gels were dried and autoradiographed to locate the positionsof the labelled proteins. By comparing -22the patterns from uninduced and induced cells, proteins synthesised under the control of the tryptophan promoter could be identified.

Figure 4 of the accompanying drawings depicts an autoradiograph of a 12.5% acrylamide: 0.1% SDS gel 14 on which are separated C-labelled proteins synthesised in E. coli cells carrying recombinant pWT 121. (asp-phe)n 14 plasmids. Track 1 showns proteins labelled with Camino acids in the presence of 40 pg/ml of L-tryptophan, i.e. any genes inserted at the Hind III site will be repressed and no protein should be produced. Track 2 14 shows proteins labelled with C-amino acids in the absence of L-tryptophan and in the presence of 5 ytg/ml of 6-indole acrylic acid, i.e. inserted genes will be fully induced and should express the protein coded for by the insert. (The protein which appears strongly in track 2 has been shown to be a repeating polymer of (asp-phe).) Tracks 3 and 4 correspond to tracks 1 and 2, respectively, except that twice the amount of protein was used. The standard protein molecular weights shown on the right of the illustration are those of a standard mixture of protein obtained from the Radiochemical Centre, Amersham, Buckinghamshire, England.

Isolation of labelled proteins from gels; particular bands of protein were isolated from acrylamide gels by separating the protein lysate in a series of —23parallel tracks. One of these tracks was cut from the gel with a scapel and stained with Coomassie Brilliant Blue R (Gurr, Searle Diagnostics) while the rest of the gel was soaked for 20 minutes in 15% v/v glycerol and stored at -70°C. The stained gel slice was dried and autoradiographed for from 24 to 48 hours to define the position of the labelled bands. This enabled the relevant portion of the frozen gel to be excised. The gel slices were broken up by forcing them through a 1 ml disposable syringe with no needle and the labelled protein eluted with 10 mM triethylammonlum bicarbonate pH 7.5 for 24 hours. Gel fragments were removed by centrifugation and the supernatant dialysed versus 10 mM triethylammonlum bicarbonate. From 50 to 75% recoveries of labelled protein bands were obtained in this way.

Enzyme digests: Crude cell lysates or isolated bands from acrylamide gels were digested either with proteolytic enzymes in solution at concentrations of from 1 to 10 mg/ml, or with equivalent amounts of enzyme immobilised on a solid support, for periods up to 72 hours. 10 mM triethylammonlum bicarbonate was 33 -24used for digests in the pH range 7-8, while 10 mM glycine/sodium hydroxide buffers were used up to pH .5.

Thin Layer chromatography: Enzyme digests of labelled proteins were separated on silica gel TLC plates (obtainable fran Merck) with concentration zone (20 x 20 cm), using either n-butanol:acetic acid:water (8:2:2 by volume) or n-propanol:conc. (0.880) ammonia (7:3 by volume) as solvent. After development, the plates were dried and autoradiographed using Kodak Kodirex X-ray film (obtainable frcm Kodak) to detect the labelled peptide products. Digests with chymotrypsin (E.C. 3.4.4,5) or subtilisin (E.C. 3.4.4.16) immobilised on a solid support or with subtilisin or proteinase K (E.C. 3.4.21.14) in soluble form all gave a mixture of products. Among these in each case was a compound which co-chromatographed with authentic aspartylphenylalanine. (Figure 5 of the accompanying drawings depicts an autoradiograph of a silica thin layer chromatography plate on which are separated the products of enzymic digestion of (asp-phe)n protein.

C-labelled (asp-phe)n protein isolated from a 12.5% acrylamide: 0.1% SDS gel was digested for 16 hours at 37°C with subtilisin immobilised on a solid support.

The thin layer chromatography plate was developed with n-propanol:0.880 ammonia (7:3 v/v). The arrows denote the positions of markers of authentic (aspphe) and (phe-asp).) This compound, when recovered -25from the TLC plate and hydrolysed in hydrochloric acid yielded only aspartic acid and phenylalanine.

Hydrolysis of induced protein to produce L-aspartlc acid and L-phenylalanine C-labelled (asp-phe)n protein isolated from an acrylamide gel, or a crude cell lysate prepared by 14 lysing induced C-labelled E. coli cells carrying pWT 121/(asp-phe) plasmids in a buffer containing 5% v/v fi -mercaptoethanol, 3% w/v SDS and 65 mM Tris.-Cl pH 8 was used for hydrolysis. A lysate made from 1 ml of an -26induced cell culture, or the isolated induced protein from a similar volume of culture was sealed in a tube with 1 ml of 6 M hydrochloric acid and heated to 110°C for 16 hrs. After this time, the tube was opened and the contents removed and evaporated to dryness.

Examination of the hydrolysate by thin layer 14 chromatography showed the presence of C-labelled L-aspartic acid and L-phenylalanine, together with smaller amounts of other amino acids.

EXAMPLE 2 A polymer (asp-phe) may be cleaved by means of an enzyme, such as chymotrypsin or subtilisin, which cuts at aromatic amino acids. Alternatively, a polymer (asp-phe-lys-lys) may be produced which is cleavable by trypsin or by an enzyme of similar specificity or by a combination of enzymes to give the dipeptide aspphe. For example, such cleavage at paired basic amino acid residues is known to be involved in the process by which hormone precursors are cleaved to active species, e.g. the corticotropin-g-lipotropin-MSHenkephalin system (Nakanishi, et al, Nature, (1979), 278, 423-427).

A gene for such a repeating tetrapeptide may be produced by the joining of four chemically synthesised dodecanucleotides:(1) A.A.G.A.T.T.T.C.A.A.A.A (2) A.G.G.A.C.T.T.T.A.A.G.A -27(3) A.G.r.C.C.T.T.T.T.T.C.A (4) Λ.Λ. T. C. Τ,Τ. T. C. T.T. A. A to form a repeating structure :asp phe lys lys asp phe lys lys asp phe ' A.A.G.A.T.T.T.C.A.A.A.A.A.G.G.A.C.T.T.T.A.A.G.A.A.A.G.A.T.T.T.T, (1) (2) (1) EcoRI' 3' T.T.C.T.A.A.A.G.T.T.T.T.T.C.C.T.G.A.A.A.T.T.C.T.T.T.C.T.A.A.A.A.5’ -If___If__i\_ (4) (3) (4) (3) This entails the use of two codons for each of the amino acids: G.A.T and G.A.C for asp; T.T.T and T.T.C for phe: and A.A.A and A.A.G for lys. These are all acceptable in E. coli. The only restriction enzyme recognition site within this polymeric gene in that for EcoRI' (A.G.A T.T.T).

Repair of the above polymeric gene using E. coli DNA polymerase I gives a molecule which reads in the correct reading frame on blunt end ligation into the Hin d III site of the plasmid pWT 111, for example.

The experimental procedure is as described in Example 1. The required polypeptide obtained on induction may be cleaved using trypsin, for example, and the aspphe dipeptide isolated.

EXAMPLE 3 Small quantities of the tripeptide Pyro-glu-hisgly have the ability to elicit an anorexic response in animals, i.e. it causes a reduction in food intake, and -28is therefore of interest in the control of appetite in humans. (0. Trygstad, et al, Acta Endocrinol, 89, 196-208, 1978).

A polymer of the form (glu-his-gly-lys-lys) would be cleaved using trypsin, for example, to yield the tripeptide glu-his-gly. Glutamic acid may be converted to pyroglutamic acid by the action of heat, see, for example, U.S. Patent No. 2,528,267.

Such a repeating polymer is coded for by a 10 repeating gene composed of four chemically synthesised oligonucleotides, two tetradecanucleotides and two hexadecanucleotides: (1) A.G.G.A.A.C.A.C.G.G.T.A.A.G. (2) A.A.A.G.A.G.C.A.T.G.G.C.A.A.A.A (3) C.T.C.T.T.T.C.T.T.A.C.C.G.T. (4) G.T.T.C.C.T.T.T.T.T.G.C.C.A.T.G These oligonucleotides may be phosphorylated and joined by ligation, using the techniques described above, to give a structure:20 glu his gly lys lys glu his gly lys ' A.G.G.A.A.C.A.C.G.G.T.A.A.G.A.A.A.G.A.G.C.A.T.G.G.C.A.A.A.(1) (2) 3’ T.C.C.T.T.G.T.G.C.C.A.T.T.C.T.T.T.C.T.C.G.T.A.C.C.G.T.T.T. >1. - - . ... ..M (4) (3) (4) lys glu his A.A.G.G.A.A.C.A.C. 3' (1) T.T.C.C.T.T.G.T.G. 5' (3) -2910 Two codons are used for each amino acid: G.A.A and G.A.G for glutamic acid; C.A.C and C.A.T for histidine; G.G.T and G.G.C for glycine; and A.A.A and A.A.G for lysine. All of these codons are acceptable in E. coli. There are no restriction enzyme recognition sites in this gene. The experimental procedure is as described in Example 1. This gene reads in the correct reading frame when blunt end ligated into the Hin d III site of the plasmid pWT 111, for example.

EXAMPLE 4 The pentapeptide arg-lys-asp-val-tyr is an analogue of the hormone thymopoietin, which has activity in inducing the differentiation of pro-thymocytes to thymocytes, (Goldstein, et al, Science, (1979), 204, 1309-1310). It has pharmaceutical application in the treatment of thymic disorders.

A repeating gene coding for a polymer of this thymic hormone analogue may be constructed from four chemically synthesised oligonucleotides, two tetradecanucleotides and two hexadecanucleotides :(1) A.C.C.G.T.a.A.A.G.A.T.G.T.T.T.A (2) C.C.G.A.A.A.G.G.A.T.G.T.C.T (3) T.T.T.C.G.G.T.A.A.A.C.A.T.C.T.T. (4) T.A.C.G.G.T.A.G.A.C.A.T.C.C These oligonucleotides may be phosphorylated and joined by ligation to give a structure:50833 -30arg lys asp val tyr arg lys asp val ' A.C.C.G.T.A.A.A.G.A.T.G.T.T.T.A.C.C.G.A.A.A.G.G.A.T.G.T.C.T.(1) (2) Acc I 3' T. G. G. C. A. T. T. T. C. T. A. C. A. A. A. T. G .G. C. T. T.T. C. C. T. A.C. A.G. A. — ι-n ,.. (3) (4) tyr A.C.C.G.T.A. 3' (1) T.G.G.C.A.T. 5' In this structure single codons are used for tyrosine (T.A.C) and aspartic acid (G.A.T), while two codons are used for arginine (C.G.T and C.G.A), lysine (A.A.A and A.A.G) and valine (G.T.T and G.T.C,. There is a single restriction enzyme recognition site within the gene for the enzyme Acc I. (G.T.C.T.A.C) this site repeats every 30 base pairs. This gene is designed to be read in the correct reading frame when inserted by blunt end ligation into the Hin d III site of the plasmid pWT 111, for example. The experimental procedure is as li’ desciilvd in ixumpic 1.

EXAMPLE 5 The enkephalins are naturally occurring peptides, found in the human brain, which are said to have a role as pain-killers. They are therefore of considerable pharmaceutical interest. There are two compounds known, met-enkephalin, which has the sequence (try-gly-glyphe-met) and leu-enkephalin, wherein the terminal 5083 -31methionine is replaced by leucine. This example relates to met enkephalin, but a similar approach is applicable to leu-enkephalin. A polymer of (tyr-gly-gly-phe-met-lys-lys) may be cleaved by trypsin, for example, to give the required pentapeptide. Such a polymer is specified by a repeating gene constructed from six chemically synthesised oligonucleotides, two octadecanucleotides and four dodecanucleotides:10 (1) G.T.A.T.G.G.T.G.G.A.T.T.T.A.T.G.A.A. (2) G.A.A.A.T.A.C.G.G.A.G.G. (3) C.T.T.T.A.T.G.A.A.A.A.A. (4) T.A.T.T.T.C.T.T.C.A.T.A.A.A.T.C.C.A. (5) A.T.A.A.A.G.C.C.T.C.C.G. (6) C.C.A.T.A.C.T.T.T.T.T.C.

These oligonucleotides may be phosphorylated and joined by ligation in one of two ways. Either all six oligonucleotides may be mixed and ligated to give the required polymer in one step. Alternatively, oligonucleotides 1, 2 and 4 and oligonucleotides 3, 5 and 6 may be ligated separately to give blocks which may then be mixed and ligated to produce the same polymer, having the structure :tyr gly gly phe met lys lys tyr gly gly ' G.T.A.T.G.G.T.G.G.A.T.T.T.A.T.G.A.A.G.A.A.A.T.A.C.G.G.A.G.G.C. . ___n. . -------------------- -Ji— (1) Mbo II, EcoRI' (2) Mnl I f.....' 3' C.A.T.A.C.C.A.C.C.T.A.A.A.T.A.C.T.T.C.T.T.T.A.T.G.C.C.T.C.C.G. (6) (4) (5) 8 33 -3215 phe met lys T.T.T.A.T.G.A.A.A.A.A. 3' (3) A.A.A.T.A.C.T.T.T.T.T.C.A.T.A.C.C. 5’ ·«. -- .. * (6) In this structure, A.T.G is used to code for methionine and T.T.T to code for phenylalanine, multiple codons are used for the other amino acids: tyrosine (T.A.T and T.A.C); glycine (G.G.T, G.G.A and G.G.C) and 10 lysine (A.A.A and A.A.G). There are single recognition sites within the gene for the enzymes Mnl I (C.C.T.C), Mbo II (G.A.A.G.A) and EcoRI' (G.G.A.T.T.T) as shown above. These sites repeat every 42 bases. The gene is designed to be read in the correct reading frame when inserted by blunt end ligation into the Hin d III site of the plasmid pWT 121, for example.

Claims

1. A synthetic gene which comprises, in the coding strand, codons for the amino acids of a desired peptide in tandem repeat.

2. A synthetic gene as claimed in claim 1 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings.

3. A process for the production of a synthetic gene as claimed in claim 1 which comprises the self-assembly and ligation and optionally repair of a plurality of appropriate oligonucleotides, the assembly to give the repeating character being achieved by self-complementary overlapping ends at the termini of the group of oligonucleotides which, when assembled, comprises the repeating unit of the gene.

4. A process as claimed in claim 3 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings.

5. A synthetic gene as claimed in claim 1 when produced by a process as claimed in claim 3 or claim 4.

6. A plasmid vector which comprises inserted therein at an insertion site adjacent to a bacterial promoter and downstream from a prokaryotic ribosome binding site a synthetic gene as claimed in any of claims 1,2 or 5 such that the gene is under bacterial promoter control. -347. A plasmid vector as claimed in claim 6 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings . 5 8. A process for the production of a plasmid vector as claimed in claim 6 which comprises inserting a synthetic gene as claimed in any of claims 1,2 or 5 at the insertion site thereof.

7. 9. A process as claimed in claim 8 substantially

8. 10 as herein described with particular reference to any of the Examples and/or the accompanying drawings. 10. A plasmid vector as claimed in claim 6 when produced by a process as claimed in claim 8 or claim 9.

9. 11. A microbial cell which has been transformed by having 15 inserted therein a plasmid vector as claimed in any of claims 6, 7 or 10.

10. 12 . A microbial cell as claimed in claim 11 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 20

11. 13. A process for the transformation of a microbial cell which comprises inserting therein a plasmid vector as claimed in any of claims 6, 7 or 10.

12. 14. A process as claimed in claim 13 substantially as herein described with particular reference to any 25 of the Examples and/or the accompanying drawings. -3515. A process for the expression of a peptide which comprises culturing a microbial cell as claimed in claim 11 or claim 12. 16. A process as claimed in claim 15 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 17. A peptide when expressed by a process as claimed in claim 15 or claim 16. 18. A synthetic gene as claimed in claim 1 for the expression of the repeating polypeptides (aspartylphenylalanine) wherein n represents an integer which is at least 2, which comprises a double-stranded DNA molecule comprising, in the coding strand, at least two nucleotide sequences coding for the expression of aspartic acid and, alternating therewith, at least two nucleotide sequences coding for the expression of phenylalanine and, in the other strand, repeating and alternating nucleotide sequences complementary to those coding for aspartic acid and phenylalanine. 19. A synthetic gene as claimed in claim 18 having the structure: 5' pT-T-T-C-G-A-C-T-T-C-G-A 3' 3' G-A-A-G-C-T-A-A-A-G-C-Tp 5' 2o. A synthetic gene as claimed in claim 18 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. -3621. A process as claimed in claim 3 for the production of a synthetic gene as claimed in claim 18 which comprises providing a first dodecanucleotide sequence coding for aspartyl-phenylalanine by linking together appropriate 5 nucleotide monomers, providing a second dodecanucleotide sequence complementary to the first sequence, phosphorylating both dodecanucleotides using polynucleotide kinase and mixing the first and second sequences together to form a double-stranded DNA structure. 10 22. A process as claimed in claim 21 in which the double-stranded structure is polymerised by incubating with - DNA ligase. 23. A process as claimed in claim 21 substantially as herein described with particular reference to any of

13. 15 the Examples and/or the accompanying drawings. 24. A synthetic gene as claimed in claim 18 when produced by a process as claimed in any of claims 21 to 23. 25. A plasmid vector as claimed in claim 6 which is pWT 121 which comprises inserted therein at the Hin d III 2o site thereof a synthetic gene as claimed in any of claims 18 to 20 or 24. 26. A plasmid vector as claimed in claim 25 substantially as herein described with particular reference to any of the Examples and/or the accompanying 25 drawings. 27. A process as claimed in claim 8 for the production of a plasmid vector as claimed in claim 25 which comprises inserting a synthetic gene as claimed in any of claims -3718 to 20 or 24 at the Hin d III site of pWT 121. 28. A process as claimed in claim 27 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 29. A plasmid vector as claimed in claim 25 when produced by a process as claimed in claim 27 or claim 28. 30. A micrcbial cell as claimed in claim 11 which is an E coli HB 101 cell which has been transformed by having inserted therein a plasmid vector as claimed in any of claims 25 26 or 29. 31. A micrcbial cell as claimed in claim 30 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 32. A process as claimed in claim 13 for the transformation of a micrcbial cell which is an E coli HB 101 cell which comprises inserting therein a plasmid vector as claimed in any of claims 25, 26 or 29. 33. A process as claimed in claim 32 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 34. A micrcbial cell as claimed in claim 30 when transformed by a process as claimed in claim 32 or claim 33. 35. A process as claimed in claim 15 for the expression of a peptide which comprises culturing a cell as claimed in any of claims 30, 31 or 34. 36. A process as claimed in claim 35 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. -3850833 VI. λ peptide when expressed by a process as claimed in claim V> nr claim 36. 38. A peptide as claimed in claim 37 which is (aspartyl-phenylalanine) wherein n represents an integer 5 which is at least 2. 39. A process as claimed in claim 35 for the expression of (aspartyl-phenylalanine) which comprises inserting a synthetic gene as claimed in any of claims

14. 18 to 20 or 24 into an insertion site of a plasmid vector 10 reading in the correct phase for the inserted gene, the insertion site being adjacent to a bacterial promoter and downstream from a prokaryotic ribosome binding site, transforming competent microbial cells using the resulting plasmid vector, culturing the transformed microbial cells 15 and harvesting the expressed peptide. 40. A process as claimed in claim 39 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 41. (Aspartyl-phenylalanine) when expressed by a 20 process as claimed in claim 39 or claim 40. 42. A process for the production of aspartylphenylalanine methyl ester which comprises subjecting (aspartyl-phenylalanine) as claimed in claim 38 or claim 41 to enzymatic cleavage using an enzyme specific 25 for amino acids having aromatic side chains to obtain aspartyl-phenylalanine and methylating the product. 43. A process as claimed in claim 42 in which the enzyme used is chymotrypsin, subtilisin or proteinase K. -3944. A process as claimed in claim 42 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings. 45. Aspartyl-phenylalanine methyl ester when 5 produced by a process as claimed in any of claims 42 to 44. 46. A process for the production of L-phenylalanine or L-aspartic acid which comprises subjecting (aspartylphenylalanine) as claimed in claim 38 or claim 41 to 10 acid or enzymatic hydrolysis and separating from the hydrolysate the desired product. 47. A process as claimed in claim 46 in which the acid hydrolysing agent is hydrochloric acid. 48. A process as claimed in claim 46 in which the 15 enzymatic hydrolysing agent is carboxypeptidase, aminopeptidase or a non-specific endopeptidase. 49. A process as claimed in claim 46 substantially as herein described with particular reference to any of the Examples and/or the accompanying drawings.

15. 20 50. L-phenylalanine or L-aspartic acid when produced by a process as claimed in any of claims 46 to 49.