FR2479259A1 - RECOMBINANT DNA TECHNIQUES - Google Patents

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

 The present invention relates to recombinant DNA techniques.

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

The present invention also relates to such a synthetic gene and the preparation thereof
Peptide synthesis by chemical means is a laborious process that becomes progressively less efficient with increasing peptide length.

On the other hand, the preparation of peptides by microorganisms during a fermentation process does not vary eficiency or eficiency as a function of the length of the product peptide. However, in the organisms used in the implementation of recombinant DNA methods, the short peptides are metabolically unstable. For example, it is known that the peptide called somatostatin, which consists of 14 amino acids, is not stable when directly produced in E. coli. Stability is achieved in this case by binding the peptide to an E. normal coli and then cleaving this fusion product. (Itakura K. et al., Science, 198, 1056 - 1063 (1977)).

In the process according to the present invention, stability and increased yield are achieved by the use of a synthetic gene containing multiple repeats of the desired small peptide coding sequence. The product is, therefore, a large molecule that is metabolically more stable in
E. coli as the small peptide. Subsequent cleavage of the large molecule generates the smaller peptide that it contains and with a higher molar yield than would be obtained if the small peptide had been directly prepared using a monomeric gene, even if the small peptide was metabolically stable.

 According to one of its embodiments, 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 repetition.

 By the term "tandem repeat" is meant herein the repetition of the codon sequence in a linear progression along the DNA molecule, with the same polarity and without interruption.

 The complementary or noncoding strand of the synthetic gene contains a sequence specified by the sequence of the coding strand, in accordance with accepted rules of nucleic acid base pairing.

 According to another of its embodiments, the present invention relates to a process for the preparation of such a synthetic gene, characterized in that it comprises the self-assembly and the binding and the possible re-pairing of a multiplicity of suitable oligonucleotides, the assembly for obtaining the recurring character being obtained by self-complementary overlapping ends at the ends of the oligonucleotide group which, in the assembled state, comprises the repeating unit of the gene.

 According to another of its embodiments, the present invention also relates to a plasmid vector, characterized in that it has been inserted into an insertion site close to a bacterial promoter and downstream from a prokaryotic ribosome binding, a synthetic gene as described above, so that the gene is under control of a bacterial promoter.

 According to another of its embodiments, the present invention relates to a cell which has been transformed by the insertion of a plasmid vector as described above.

 According to yet another of its embodiments, the present invention also relates to a method for the expression of a peptide, characterized in that it comprises the culture of such a cell.

 As indicated above, the synthetic gene strands are prepared from a series of short oligonucleotides which are themselves prepared by mutually linking the desired nucleotides by the use of known methods, in a known manner. specified order, by the amino acid codons of the desired peptide and following the rules described in more detail below.

The oligonucleotides of the coding strand and of the complementary strand of the gene form an overlapping game corresponding to the following general structure

<tb> 5 '<SEP> a, <SEP> b <SEP> c <SEP> d <SEP> e <SEP> f <SEP>3'<SEP>
<tb><SEP> I <SEP> I <SEP>
<tb> 3t <SEP>! <SEP> I
<tb><SEP> b <SEP> c <SEP> d <SEP> e <SEP> f <SEP> a
<tb><SEP> c <SEP> d <SEP>, <SEP> d <SEP> 1 <SEP> e <SEP> f <SEP> a <SEP>
<Tb>
The structure shown in this illustration consists of six oligonucleotides, three in each strand.However, the structure can be assembled from any number of oligonucleotide pairs from an upward pair, provided that
(1) the paired regions designated a1 and a2, b1 and b2, c1 and c2, ... contain nucleotide base sequences which are complementary in the pair, but not sequences of any other pair, 1 each e 2 1 regions a, b, 2 1
(2) ... each of these regions a, a, b, b, c c2 ... contains at least four nucleotide bases and
(3) the sum of the nucleotide bases in each strand which, in the assembled state, optionally comprises the recurring length of the polymer, being divisible by three.

 When a set of oligonucleotides of this character is mixed, the complementary regions of the oligonucleotide pairs hybridize to generate high molecular weight structures in which the oligonucleotide sequences (a1-f1 in the above illustration ) In the subsequent process, it is desirable to maximize the number of polymers that have, in the sequence, at their 5 'ends. This is achieved by mixing oligonucleotides a1 b1, c1 d1 e1 1 b2 c2 and of the above illustration in equimolar amounts and subsequently adding the oligonucleotide f2 a2 in an amount less than the equimolar amount.Since the supplements of a1 and f1 are present in sub-molar quantities, most polymers terminate in a and fi. In the general case, the 5'-terminal oligonucleotide of the complementary set adds to the polymerization mixture in an amount which is less than the equimolar proportion compared to other oligonucleotides which are all present in equimolar amounts.

 Chain "cuts", where adjacent oligonucleotides in the channe juxtapose, can be joined by the use of the enzyme autest DNA ligase (EC 6.5.1.1), which is a known method.

 The molecules thus prepared will have single-stranded ends. These can be converted into the so-called "blunt end" form by using the enzyme DNA polymerase I (EC 2.7.7.7), using a known method.

 The molecules thus obtained can be cloned into competent microbial cells, in particular E. coli, using a desired vector. More specifically, a plasmid vector selected from the series called npWT (see, for example, Tacon et al., Molec Gen. G-et., 177, 427, 1980) may be used, the choice of vector being made such that when the synthetic gene is inserted in the correct orientation, it is read in the desired translation reading frame.

Figure 1 of the accompanying drawings illustrates a scheme for producing a recombinant plasmid (pWT 121 (asp-phe) n) which contains the recurring gene for (asp-phe) n in the correct DNA phasing for expression of a protein at (asp-phe) n. The plasmid DNA, shown at the top of the diagram, is cut with the restriction enzyme
Hind III and is then reappropriated with the DNA polymerase I of E. coli. coli to generate a blunt ended molecule.

Polymer coding for (asp-phe) n, shown at the bottom of the scheme, has its initially projecting 5 'end similarly re-paired to generate another blunt ended molecule. The union of these two blunt end species with T4 DNA ligase generates the recombinant DNA, shown in the center of the scheme, with the correct DNA phasing for (asp-phe) n expression.

 The cloning of this gene, the choice of bacterial colonies, the detection of the polypeptide produced by the expression of the gene and the extraction and purification of the polypeptide can all be carried out using known methods.

 This polypeptide may then be further cleaved either in an enzyme manner using specific enzymes or chemically, for example by the use of cyanogen bromide to cleave or cleave the polypeptide chains to methionine residues.

 As will be understood by those skilled in the art, the present invention finds numerous applications. Among others, the present invention can be applied to the peptide hormones oxytocin, vasopressin and somatostatin, to enkephalins (opiate pentapeptides) and peptides that regulate appetite.

 For purposes of illustration, a particular embodiment of the present invention will now be described in more detail with particular reference to the sweetener product "Aspartame".

 Aspartame is a low-calorie artificial sweetener that can be described chemically as the methyl ester of aspartylphenyl alanine. Aspartame is commonly produced by a multi-step chemical synthesis.

 Due to its use as a sweetener, the total consumption of Aspartame is potentially very important and there is therefore considerable interest in the development of preparations through which this material can be conveniently obtained on a large scale. .

 It has been found that Aspartame can be prepared by carrying out a potentially large-scale method based on recombinant DNA techniques, in accordance with which a synthetic gene coding or code for aspartyl-phenylalanine is inserted. ) n in a cloning vector having a bacterial promoter controllable upstream of and substantially adjacent to the insertion site and the vector can be used to transform bacterial cells from which the expressed polypeptide can be obtained, which polypeptide can then be split so as to obtain aspartyl-phenylalanine and, therefore, 1-aspartame.

 Therefore, according to another of its embodiments, the invention also relates to a synthetic gene for the expression of the recurring polypeptide (aspartyl-phenylalanine) n, characterized in that it comprises a double DNA molecule. strand or double-strand comprising, in the coding strand, at least two trimers of nucleotides coding for the expression of aspartic acid (Asp) and, alternatively with these, at least two trimers of nucleotides coding for the expression of phenylalanine (Phe) and, in the other strand, recurrent and alternating nucleotide sequences, complementary to the coding sequences (Phe) and (Asp).

The coding strand of the synthetic gene, i.e. that which codes for the recurrent polypeptide (Asp-Phe) n, can therefore be represented as a coding
5 '- (Asp - Phe - Asp - Phe) - 3' where n represents an integer, for example up to several hundred.

avec un brin complémentaire comportant la séquence
3' -A-A-A-G-C-T-G-A-A-G-C -T-A-A-A-G-C-T-G-A-A- 5'
Dans ce cas, on utilise le (T-T-C) comme séquence codant pour la phénylalanine et les deux codons (G-A-C) et (G-A-T) alternent pour l'acide aspartique.There are two possible codons for Phe, namely (TTT) and (TTC) and two possible codons for
Asp, namely (GAT) and (GAC) and these codons can be used in any permutation with corresponding complementary sequences in the other strand. For example, the coding strand can be a nucleotide sequence as follows

with a complementary strand with the sequence
3 '-AAAGCTGAAGC -TAAAGCTGAA- 5'
In this case, the (TTC) is used as the coding sequence for phenylalanine and the two codons (GAC) and (GAT) alternate for aspartic acid.

 Other permutations and possible combinations of the 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 can be prepared by the polymerization of a double stranded DNA fragment consisting of two dodecanucleotide strands, one comprising the sequence for the expression of (Asp-Phe) 2, the encoder strand and the other being complementary thereto, namely the complementary strand,
Therefore, according to another of its embodiments, the present invention relates to a method characterized in that a first dodecanucleotide sequence encoding aspartyl-phenylalanine is made by mutually binding appropriate monomeric nucleotides, performing a second dodecanucleotide sequence complementary to the first sequence, phosphorylating the two dodecanucleotides using T4-polynucleotide kinase and mutually mixing the first and second sequences so as to form a double-stranded DNA structure.

The dodedanucleotide strands can be each synthesized from the respective monomers which are protected by the blocking groups in a known manner and linked by conventional phosphotriester methodology (see, for example, Hsiung et al., Nucleic Acids
Research, 6, 1371-1355 (1979)), the dodecamers thus obtained being unblocked and subsequently purified. After purification and phosphorylation using T4polynucleotide kinase (EC 2.7.1.78), the coding and complementary strands are mutually mixed resulting in the formation of double-stranded structures by hydrogen bonding. dodecamer coding for (Asp-Phe) 2 for the coding strand and a corresponding dodecanucleotide complementary strand, extremely stable double-stranded structures were obtained resulting from the overlapping of six strand bases, which, for the example previously given, are as follows
5 'pT-TTCGACTTCGA 3'
3 'GAAGCTAAAGC-Tp 5'
The linear polymerization of the aforementioned double-stranded structures is obtained by incubation with T4-DNA ligase (EC 6.5.1.1) to generate long polymers of stochastic length. After separation of the unbound material, it is found that a range of polymers containing from 2 to 500 and more than 500 repeats of the base units is obtained.

 The present invention also relates to a process for the expression of (aspartyl-phenylalanine) n ', characterized in that a synthetic gene as described above is inserted 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 of a prokaryotic ribosome binding site, in that competent microbial cells are transformed using the plasmid vector thus obtained, in that the transformed cells are cultured and the expressed peptide is harvested.

In addition, the polymer product is considered new and the scope of the invention also extends.

 The present invention also relates to a process for the preparation of Aspartame (aspartyl-phenylalanine methyl ester), characterized in that the abovementioned synthetic gene is inserted into a plasmid cloning vector, for example pWT 121 , designed to read in the correct phase for expression of the inserted gene and having a bacterial promoter upstream of and adjacent to the insertion site. The plasmid vector can be used to transform E. coli HB 101 cells. (Asp-Phe) n expressed can be harvested and subjected to enzymatic cleavage using an enzyme specific for amino acids with aromatic side channels, such as subtilisin (EC 3.4.4.16), chymotrypsin (EC 3.4.4.5) or proteinase K (EC 3.4.21.14), so as to obtain aspartyl-phenylalanine which is methylated to generate Aspartame. The enzyme used can be in a soluble form or, preferably, it can be immobilized on a solid support. The methylation can be carried out using conventional chemical means, or it can be carried out by a reaction. exchange with the enzyme in the presence of methanol or by direct enzymatic methanolysis of the polymer.

 It should be understood that, given the triplet nature of the genetic code, the gene can be read in any of three phases and that it is therefore essential for a gene to use, as a vector of plasmid, a plasmid vector that provides translation in the correct frame or reading frame.

 It has been discovered that a preferred plasmid vector to be used for carrying out the method according to the invention is a plasmid vector selected from the above-mentioned "pWT" series.

Basically, plasmids of the p1T series comprise a Hind III restriction site (EC 3.1.23.21) adjacent to a tryptophan promoter of E. coli. coli clone
By the restriction to Hind III and the insertion of Hind III binders, it is possible to construct, in the pWT series, plasmids capable of translating into all frames or reading frames, these plasmids being pWT 111, pWT 121 and pWT 131 as described in the above-mentioned reference.

Transcription and translation of the DNA inserted into the pA series plasmids is under the direct and powerful control of tryptophan; thus, in the presence of tryptophan, the operon is repressed whereas in the absence of tryptophan it is derepredivated.
The plasmids of the p1E series also possess, in the region immediately following the Hind III site, the gene for tetracycline resistance, so that after the insertion of the DNA, transcription and translation can can easily be confirmed, since when it has occurred, the resistance to tetracycline is maintained.

In a preferred embodiment of the method according to the invention, the synthetic gene is therefore inserted into the appropriate plasmid of the pWT series, ie the plasmid called "pWT 121. This plasmid is schematically illustrated in FIG. drawings and has the following characteristics:
molecular length of 4837 bp; an Hpa I site (EC 3.1.23.23); a Hind III site at 206 bp from the Hpa I site; a Bam HI site (EC 3.1.23.6) at 353 bp of the Hind III site; a Sal I site (EC 3.1.23.37) at 275 bp from the Bam HI site; a Pst I site (ECD1.23.31) at 2958 bp from the Sal i site and at 1045 bp from the Hpa I site; the gene for tetracycline resistance extending from the region of the Hpa I site to beyond the Sal I site, the gene for ampicillin resistance being in the region of the Pst 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 Hind III sites.

The synthetic Aspartame gene according to the present invention was cloned into pWT 121 as follows
Plasmid pWT 121 was restriction-restricted with the Hind III enzyme to obtain a linear molecule which was then treated with DNA polymerase and all four deoxyribonucleoside triphosphates to produce "ends". The blunt-ended molecule was then treated with bacterial alkaline phosphatase (EC 3.-I.3.1) to remove the 5'-phosphates, and the plasmid was prepared for the preparation. insertion into the synthetic gene which has been previously treated as follows
The synthetic gene was treated with E. coli DNA polymerase. coli and all four deoxyribonucleoside triphosphates so as to produce blunt ends, and the "reapplied" genes were separated from the enzyme and small molecules.

 The blunt-ended vector DNA and the blunt-ended gene were mixed in a blunt-ended fashion.

port ranging from 1: 1 to 1: 2 and mutually bound with high concentrations of T4-DNA ligase, so as to obtain the plasmid series "pWT 121 / (asp-phe) n".

The sizes of the cloned inserted entities, determined by restriction enzyme digestion, were found to be other than 60 to 900 bp (i.e., 5-75 repeats of the dodecanucleotide unit).

 Plasmids pWT 121 / (asp-phe) n, prepared as described above, were used to transform E. coli cells. coli, in a known manner, for example, by the exposure of cells treated with calcium chloride to plasmid pWT / (asp-phe) n. The transformed cells are cultured in a manner known in a medium containing no tryptophan and preferably containing the inducer 8-indoleacrylic acid is expressed a protein consisting essentially of (asp-phe) n which after enzymatic cleavage and methylation, produces the desired methyl aspartyl-phenylalanine methyl ester, namely Aspartame.

 As mentioned above, Aspartame is conventionally obtained by a multi-step chemical synthesis. L-Phenylalanine is used as an intermediate for carrying out this conventional synthesis. The present invention also relates to the preparation of L-phenylalanine which is of interest for this conventional synthesis. When phenylalanine is produced by means of chemical synthesis, this product is obtained in the form of racemic which. must be solved before the Isomer L can be obtained and this resolution is an additional step and storyteller of the implementation of the process in question.

When it is obtained by carrying out the process according to the invention, phenylalanine is obtained directly in the form of the L isomer.

 Therefore, the present invention also relates to a process for the preparation of L-phenylalanine, characterized in that the peptide (asp-phe) n isolated from a culture of E. coli cells is treated. coli carrying plasmids incorporating the synthetic Aspartame gene, an acidic or enzymatic hydrolysis agent and that the desired L-phenylalanine is separated from the hydrolyzate.

This process therefore also generates the acid
L-aspartic and the scope of the present invention also extends to such a method.

 Preferred hydrolysis agents include hydrochloric acid or carboxypeptidase (E.C. 3.4.12.x), aminopeptidase (E.C. 3.4.11.x) or aspecific endopeptidases.

 The separation of the hydrolysis products can be carried out using known methods.

 The following examples illustrate the present invention without limiting the latter.

EtEPLE 1 (asp-phe) n
Synthesis of dodecanucleotides
Both dodecanucleotides were synthesized,
TpCpGpApApApTpCpGpApApG and TpTpTpCpGpApCpTpTpCpGpA, by conventional triester methodology, as described, for example, by Hsiung, Loc cit, and in accordance with the reaction scheme illustrated in Figure 3 of the accompanying drawings wherein N represents Gis obu, T, çbz or AbZ .

The fully protected dodecanucleotides were deblocked with benzene sulfonic acid at 2/0 w / v, 0.1 M tetraethylammonium fluoride in a mixture of tetrahydrofuran, pyridine and water (8: 1: 1 by volume), then by treatment with ammonia. Unblocked dodecanucleotides were purified by high pressure liquid chromatography on "Partisil SAX" (microparticulate silica which was derived using quaternary ammonium groups (QMatman)).

Polymerization of dodecanucleotides
Each synthetic dodecanucleotide (2 μg) was phosphorylated in a reaction medium of 10 μl containing from 10 to 50 μl of 32 P-ATP, 50 mM Tris-Cl pH 7.8, 5 mM magnesium chloride , 25 mM unlabelled ATP, 10 mM mercaptoethanol and 3 units (1 unit is the amount that causes the transfer of 1 nanomole of 32 P-phosphate of Y 32 P) -ATPurslextremity 5'-hydroxy of a polynucleotide, in 30 minutes, at 370C, under normal test conditions, see, for example, Richardson,
Nucleic Acids Research, 2, 815) of T4 polynucleotide kinase (EC 2.7.1.78). After 60 minutes at 370C, the two dodecanucleotides were mixed, unlabeled ATP was added to bring ATP to 1 at the same time as 0.1 unit (1 unit is the amount that converts 100 monomoles of d (A-T) 1000 to an exonuclease III resistant form, within 30 minutes, at 300C, in normal test conditions, see, for example,
Modrich and Lehman, J. Biol Chem., 245, 3626 (1970)) of
T4 DNA ligase (EC 6.5.1.1) and the reaction mixture was incubated for 24 hours at 250C. We. ATP and unbound monomeric nucleotides were removed by descent through a column (20 cm x 0.8 cm) of Sephadex G-5011 (modified, crosslinked, particulate, superfine dextran polymer (Pharmacia)) in 50 mN sodium chloride, 10 mN Tris-Cl pH 7.5, and 0.2 O / op / v sodium dodecyl sulfate (SDS). The double-stranded polymers were concentrated by methanol precipitation. After sepa- ration, the product was found to be a mixture of polymers of stochastic length. containing from 2 to more than 500 repetitions of base units.

Cloning of the synthetic gene
(a) Matching the blunt end of the
synthetic gene
2 g of the polymer prepared as described above was incubated in a mixture of 40 l of 50 mM Tris-Cl pH 7.8 3 with 5 mN magnesium chloride, 1 mN mercaptoethanol, 0.125 mM of each of the four deoxynucleotide triphosphates and 2 units (1 unit is the amount which causes the incorporation of 10 nanomoles of nucleotide in an acid-fast form, in 30 minutes, at 37 ° C, under conditions of normal test using poly-d (A-T) as a template, see, for example, Richardson and colzas
J. Biol. Chem., 239, 222, (1964)) of E. coli DNA polymerase I (EC 2.7.7.7). The mixture was incubated for 20 minutes at 10 ° C. and the mixture was used without further purification.

(b) Preparation of the cloning vector pWT 121
The digestion of 50 μg of pWT 121 DNA was induced by a double excess of Hind III (EC 3.1.23.21).

The mixture was extracted twice with an equal volume of phenol and precipitated with ethanol.

 Re-pairing with E. coli DNA polymerase was performed. coli to generate blunt ends as described in (a) above.

 Blunt-ended DNA was then treated with bacterial alkaline phosphatase (E.C.

3.1.3.1), in order to remove terminal phosphates and to prevent the recircularization of the DNA during the subsequent bonding. The DNA was incubated for 30 minutes at 370 ° C., in the presence of a double excess of the enzyme in 10 ml. Tris-Cl pH 7.5 and SDS 0.1 / 0. The mixture was then exhaustively extracted with phenol, washed repeatedly with chloroform and then precipitated with ethanol.

(c) Blunt end bonding
The blunt ended polymeric dodecanucleotide of (a) above and the blunt ended plfT 121 DNA of (b) above were mixed in a weight ratio of 1: 1 and bound at 150C to 0.2. T4 DNA ligase unit in a 20 l mixture containing 50 mN Tris-Cl pH 7.8, 50 mM magnesium chloride, 1 mM ATP and 10 mN mercaptoethanol. After 24 hours, recombinant plasmids pWT 121 / (asp-phe) n were ready for use to transform E. coli cells. coli.

Transformation and gene expression
E.cells were transformed. coli K12 HB 101 (genotype gal, lac-, ara, pro, arg, str, rec A, rk- '
Mk-; Boyer HW and Roullard - Dussoix D., J. Mol. Biol., 41, 459-472) by carrying out the method of Katz et al., (1973) J. Bacteriol., 114, 577-591 and plated on L-agar plates supplemented with Ampicillin (100 Fg / ml).

The recombinant clones were purified by streaking so as to obtain unique colonies. Clones thus isolated were examined by colony hybridization on nitro-cellulose filters (Grunstein and Hogness (1975) Proc Nat Acad Sci USA 72, 3961-3965) using a kinase-labeled synthetic dodecanucleotide. hybridization probe. Positive single colonies were raised by colony hybridization to an A600 of 0.6 in 25 ml of M9 medium (tiller, (1972), Experiments in Molecular Genetics, Cold Spring Harbor Laboratory,
New York, page 433) supplemented with ampicillin (100 g / ml) and tryptophan (40 ug / ml). A 1 ml sample of this repressed culture was taken and labeled for 10 minutes with 5 .mu.C of 14 C-amino acids and treated for 10 minutes with 200 .mu.l of "casamino acids" at 20.degree. op / v (Difco). The cells were pelleted (10,000 xg for 10 minutes), washed repeatedly with phosphate buffered saline containing 100 μg / ml of gelatin to remove the excess of marker and the final part was lysed in 50 μl of buffer (FSB) containing 10 μg / v of glycerol, 0.01 w / v / v of bromophenol blue, 5 O / ov / S-mercaptoethanol, 3% w / v SDS and 65 mM Tris-Cl pH 8 by heating to 900C for 2 minutes.

The remainder of the initial 25 ml culture (10,000 xg for 10 minutes) was pelleted and resuspended in an equal volume of M9 medium supplemented with ampicillin (100 Ag / ml) and acid. R-indoleacrylic (5 tig / ml).

After various induction periods, 1 ml samples were taken and labeled as described above. Aliquots of 5 to 10 Al were separated from the final lysates on the 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 positions of the labeled proteins. By comparing the motifs from uninduced and induced cells, it was possible to identify the proteins synthesized under the control of the tryptophan promoter.

Figure 4 of the accompanying drawings represents an autoradiograph of a gel of 12.5% acrylamide: 0.1% SDS on which are separated C-labeled proteins synthesized in cells. of. coli carrying pWT 121 plasmids (recombinant asp-phe> n). Lane 1 shows proteins labeled with 14 C-amino acids in the presence of 40 μg / ml of L-tryptophan, i.e. any genes inserted at the Hind III site will be repressed and no protein will be produced Lane 2 shows proteins labeled with 14 C-amino acids in the absence of Ltryptophan and in the presence of 5 Fg / ml of B-indole acrylic acid, i.e. the inserted genes will be fully induced and will be able to express the protein encoded by the inserted product. (It has been shown that the protein that strongly appears on lane 2 was a 3 and 4 correspond to lanes 1 and 2 respectively, except that twice the amount of protein has been used.The molecular weights of the standard proteins shown on the right of illustration are those of a standard mixture of proteins obtained from the radi center ochimic of Amersham, Buckinghamshire,
England.

Isolation of marked rotenes from gels
Particular bands of acrylamide gel proteins have been isolated by separating the protein lysate in a series of parallel tracks. One of these gel tracks was cut with a scalpel and stained with Coomassie R brilliant blue (Gurr, Searle).
Diagnostics), while the rest of the gel was soaked for 20 minutes in O / ov / v glycerol and stored at -700C. The stained gel slice was dried and autoradiographed for 24 to 48 hours to define the position of the labeled bands. This allowed the relevant part of the frozen gel to be excised. The gel slices were broken by forcing through a disposable 1 ml needle-free syringe and the labeled proteins were eluted with 10 mM triethylammonium bicarbonate. pH 7.5 for 24 hours. The gel fragments were removed by centrifugation and the supernatant layer dialyzed against triethyl ammonium bicarbonate. In this way, recoveries of 50 to 75% of labeled protein bands were obtained.

Digestion with enzymes
Digestion of crude cell lysates or isolated bands of acrylamide gels was either using proteolytic enzymes in solution at concentrations of 1 to 10 mg / ml, or using equivalent amounts of enzyme immobilized on a solid support, for periods up to 72 hours. Triethylammonium bicarbonate 10 mE was used in the pH range of 7 to 8, while 10 mM sodium hydroxide / glycine buffers were used up to a pH of 10.5.

Thin layer chromatography
Enzyme digests were separated from labeled proteins on Merck silica gel thin layer plate plates with a concentration zone (20 x 20 cm) using either n-butanol acetic acid and water (8 = 2: 2 by volume), a mixture of n-propanol and concentrated ammonia (0.880) (7: 3 by volume), as a solvent After the development, dried plates and autoradiographed using Kodak "Kodirex" X-ray film to detect labeled peptide products.

Digestion with chymotrypsin (EsC. 3,4,4.5) or subtilisin (EC 3.4.4.16) immobilized on a solid support, or with subtilisin or proteinase K (EC 3.4.21.14) in soluble form have all given a mix of products. Among these was, in each case, a compound that co-chromatographed with authentic aspartyl-phenylalanine.

(Figure 5 of the attached drawings represents an autoradiography of a silica thin layer chromatography plate on which are separated the products of enzymatic protein digestion (asp phe) ne
The 14 C-labeled protein (asp-phe) isolated from an acrylamide gel at 12.5%: SDS at 0.1% for 16 hours at 37 ° C. was digested with subtilisin immobilized on a support. solid. The thin layer chromatographic plate was developed with a mixture of n-propanol and 0.880 ammonia (7: 3 v / v). The arrows indicate the positions of the authentic (asp-phe) and (phe-asp) markers. The compound, recovered from the thin-layer chromatography plate and hydrolysed in hydrochloric acid, only gave aspartic acid and phenylalanine.

Hydrolysis of Proteins Induced to Generate L-Asaric Acid and L-Phenylalanine
For hydrolysis, the 14C-labeled protein (aspphe) isolated from an acrylamide gel or a crude cell lysate prepared by lysing E. coli cells was used. 14C-labeled coli carrying plasmas of pWT 121 / (asp-phe) n) I in a buffer containing 5 μg / v of 8-mercaptoethanol, 30 μg / v of SDS and 65 mM of
Tris-Cl pH 8. A lysate prepared from 1 ml of an induced cell culture, or induced protein isolated from a similar volume of culture, was sealed in a tube with 1 ml of hydrochloric acid. M and the tube was heated at 1100C for 16 hours. After this time, the tube was opened and its contents removed and evaporated to dryness. Examination of the hydrolyzate by thin layer chromatography revealed the presence of L-phenylalanine and 14 C-labeled L-aspartic acid together with small amounts of other amino acids.

EXAMPLE 2
An (asp-phe) polymer can be cleaved by an enzyme, such as chymotrypsin or subtilisin, which cuts the aromatic amino acids. It is also possible to produce a polymer of (asp-phe-lys-lys) n which can be cleaved or cleaved by trypsin or by an enzyme of similar specificity or by a combination of enzymes to generate the asp-phe dipeptide. For example, such cleavage with the paired basic amino acid residues occurs, as is well known, in the process by which hormone precursors are split into active species, for example the corticotropin-lipotropin-SH system. - Enkephalin (Nakanishi et al., Nature, (1979), 278, 423-427).

A gene for such a recurrent tetrapeptide can be produced by the union of four chemically synthesized dodecanucleotides:
(1) AAGATTTCAAAA
(2) AGGACTTTAAGA
(3) AGTCCTTTTTGA
(4) AATCTTTCTTAA to form a recurrent structure

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 lilies. These are all acceptable in E. coli. The only restriction enzyme recognition site in this polymer gene is that for EcoRI (A.G.A.T.T.).

Reconciliation of the aforementioned polymer gene using XDN polymerase I dE. coli generates a molecule that reads in the correct reading frame or frame on the blunt end link in the site
Hind III plasmid pWT 111g for example
The experimental method is as described in Example 1. The necessary polypeptide obtained by induction can be further cleaved using trypsin, for example, and the dipeptide asp-phe can be isolated.

EXAMPLE 3
The tripeptide Pyro-glu-his-gly, in small quantities, has the capacity to give rise to an anorexic response in animals, that is to say it causes a reduction in the ingestion of food and that it is therefore of interest for the control of appetite in humans (Trygstad et al., Acta Endocrinol., 89, 196-208, 1978).

 A polymer of the form (glu-his-gly-lys-lys) could be cleaved using trypsin, for example, so as to generate the glu-his-gly tripeptide.

It is possible to convert glutamic acid to pyroglutamic acid under the effect of heat, and it is sufficient in this connection to consult, for example, United States Patent No. 2,528,267.

Such a recurring polymer is encoded by a repeating gene composed of at least four chemically synthesized oligonucleotides, two tetradecanucleotides and two hexadecanucleotides.
(1) AGGAACACGGTAAG

 (2) A.A.A.G.A.G.C.A.T.G.G.C.A.A.A.

 (3) C.T.C.T.T.T.T.T.T.T.A.C.C.G.T.

 (4) G.T.T.C.T.T.T.T.T.T.G.C.C.T.T.G.

These oligonucleotides can be phosphorylated and bonded together, using the techniques described above, so as to obtain a structure such as the following

Two codons are used for each amino acid
GAA and GAG for glutamic acid, CAC and CAT for lthistidine, GGT and GGC for glycine and AAA and AAG for lysine. All these codons are acceptable in E. coli. there are no restriction enzyme recognition sites in this gene. The experimental method is as described in Example 1. This gene reads into the correct frame or reading frame when blunt-ended in the Hind III site of plasmid pWT 111, for example.

EXAMPLE 4
The pentapeptide arg-lys-asp-val-tyr is an analogue of the hormone thymonoSetine, which has the activity of inducing the differentiation of pro-thymocytes into thymocytes (Goldstein et al., Science, (1979). ), 204, 1309-1510). it has a pharmaceutical application for the treatment of mood disorders.

A recurrent gene encoding a polymer of this thymic hormone analog can be constructed from four chemically synthesized oligonucleotides, namely two tetradecanucleotides and two hexadeconucleotides (1) ACCGTAAAGATGTTTA
(2) CCGAAAGGATGTCT
(3) TTTCGGTAAACATC0TT
(4) TACGGTAGACATCC
These oligonucleotides can be phosphorylated and bound together, so as to generate the following structure

In this structure unique codons are used for tyrosine (TAC) and aspartic acid (GAT), while two codons for arginine (CGT and CGA), lysine (AAA and AAG) and valine (GTT and GTC). there is a unique restriction enzyme recognition site in the gene for the Acc I enzyme (GTCTAC), this site is repeated every 30 base pairs. This gene is intended to be read in the correct frame or reading frame, when inserted by blunt end link into the site
Hind III of plasmid pWT 111, for example. The experimental method is as described in Example 1.

EXAMPLE 5
Enkephalins are natural peptides that are found in the human brain and are said to play a role as pain-killing substances.

They are therefore of considerable pharmaceutical interest. Two known compounds, metenkephalin, have the following sequence (try-glygly-phe-met) and leu enkephalin, where terminal methionine is replaced by leucine. This example is relative to met-enkephalin, but a similar approach applies to leu-enkephalin. A polymer of (tyr-gly-gly-phe-met-lys-lys) can be cleaved by trypsin, for example, so as to generate the desired pentapeptide.

Such a polymer is specified by a recursive gene constructed from six chemically synthesized oligonucleotides, namely two octadecanucleotides and four dodecanucleotides.
(1) GTATGGTGGATTTATGAA

 <2) G.A.A.A.T.A.C.G.G.A.G.G.

 (3) C.T.T.T.T.T.A.T.G.A..A.A.A.A.

 (4) T.A.T.T.T.T.T.T.T.A.T.A.A.A.A.A.A.A.A.A.A.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 can be phosphorylated and bound together in two or three ways. All of these six oligonucleotides can be mixed and bound so as to generate the polymer in a single step. But oligonucleotides 1, 2 and 4 and oligonucleotides 3, 5 and 6 can also be separately bound so as to generate blocks which can then be mixed and bound so as to generate the same polymer, corresponding to the following structure

 In this structure, ATG is used as a code for methionine and TTT-as a code for phenylalanine, multiple codons are used for the other amino acids: tyrosine (TAT and TAC), glycine (GGT, .GGA and GGC) and lysine (AAA and AA, G).

These are unique recognition sites in the gene for the enzymes Iinl I (CCTC) Mbo II (G.AXA.GA) and
EcoRi '(GGATTT) as shown above. These sites are repeated every 42 bases. This gene is intended to be read into the correct frame or reading frame when inserted by blunt end binding into the Hind III site of plasmid pAfT 121, for example.

Claims (34)

 1. Synthetic gene, characterized in that it comprises in the coding strand codons for the amino acids of a desired peptide in tandem repetition.  2. A method for producing a synthetic gene according to claim 1, characterized in that it comprises the self-assembly and the binding and the possible re-pairing of a multiplicity of appropriate oligonucleotides, the assembly to give the recurring character being obtained by autocomplementary overlapping ends at the ends of the oligonucleotide group, which, in the assembled state, comprises the repeating unit of the gene.  3. synthetic gene according to claim 1, when obtained by carrying out the process according to claim 2.  4. Plasmid vector, characterized in that it comprises, inserted at an insertion site adjacent to a bacterial promoter and downstream of a procariote ribosome binding site, a synthetic gene according to any one of claims 1 and 3, so that the gene is under the control of a bacterial promoter.  5. Process for the production of a plasmid vector according to claim 4, characterized in that a synthetic gene according to any one of claims 1 and 3 is inserted at the insertion site of the vector.  Plasmid vector according to claim 4, obtained by carrying out a process according to claim 5.  7. A cell that has been transformed by inserting a plasmid vector according to any one of claims 4 and 6.  8. A method of transforming a cell, characterized in that there is inserted a plasmid vector according to any one of claims 4 and 6.  9. A cell when it has been transformed by carrying out a process according to claim 8.  10. Process for the expression of a peptide, characterized in that culture of a cell according to one of Claims 7 and 9 is carried out.  Peptide when expressed by a method according to claim 10.  12. synthetic gene according to claim 1 for the expression of the recurring polypeptides (aspartyl-phenylalanine) n wherein n represents an integer which is equal to at least 2, characterized in that it 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 recurrent and alternating nucleotide sequences complementary to those coding for aspartic acid and phenylalanine.

 13. Synthetic gene according to claim 12, characterized in that it responds to the following structure <Tb> <tb> 3 '<SEP> 3' <SEP> G-A-G-C-T-A-A-A-G-C-Tp <SEP> 5 ' <tb> 5 '<SEP> pT-T-T-C-G-A-C-T-T-C-G-A <SEP> 3'  14. A method according to claim 2 for the production of a synthetic gene according to claim 12, characterized in that a first dodecanucleotide sequence coding for aspartyl-phenylalanine is produced by mutually binding suitable monomeric nucleotides, carrying out a second dodecanucleotide sequence complementary to the first sequence, the two dodecanucleotides are phosphorylated using T4 polynucleotide kinase and the first and second sequences are mutually mixed so as to generate a structure ("double-stranded DNA  15. Process according to claim 14, characterized in that the double-stranded structure is polymerized by incubation with T4-DNA ligase.  16. Synthetic gene according to any one of claims 12 and 13 when produced by carrying out a process according to any one of claims 14 and 15.  Plasmid vector according to claim 4, which is pWT 121, characterized in that a synthetic gene according to any one of claims 12, 13 and 16 has been inserted at the Hind III site.  The method according to claim 5 for producing a plasmid vector according to claim 17, characterized in that a synthetic gene according to any one of claims 12, 13 and 16 is inserted at the HindIII site of pWT. 121.  Plasmid vector according to claim 17, obtained by carrying out a process according to claim 18.  The cell of claim 7, which is a cell of E. coli HB 101, which has been transformed by inserting a plasmid vector according to any one of claims 17 and 19.  21. The method of claim 8 for transforming a cell that is a cell of E. coli HB 101, characterized in that a plasmid vector according to any one of claims 17 and 9 is inserted therein.  22. Cell according to claim 20, when it has been transformed by carrying out a process according to claim 21.  23. A process according to claim 10 for the expression of a peptide, characterized in that a cell is cultured according to any one of claims 20 and 22.  24. A peptide when expressed by a method according to claim 23.  25. (Aspartyl-phenylalanine) n wherein n represents an integer which is at least 2.  26. The method according to claim 23 for the expression of (aspartyl-phenylalanine) n, characterized in that a synthetic gene according to any one of claims 12, 13 and 16 is inserted 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 of a procariote ribosome binding site, in that the cells are transformed microbial competent using the plasmid vector thus obtained, in that one cultured transformed cells and in that one harvests the peptide expresses.  27. (Aspartyl-phenylalanine) n when expressed by a method according to claim 26.  28. Process for the production of the methyl ester of 11-aspartyl phenylalanine, characterized in that the (aspartyl-phenylalanine) n according to any one of claims 25 and 27 is subjected to enzymatic cleavage using a specific enzyme for amino acids having aromatic side rings, so as to obtain aspartyl-phenylalanine and the product is methylated.  29. Process according to claim 28, characterized in that the enzyme used is chymotrypsin, subtilisin or proteinase R.  30. A methyl ester of aspartyl-phenylalanine when it has been obtained by carrying out a process according to any one of claims 28 and 29.  31. Process for producing L-phenylalanine or L-aspartic acid, characterized in that the (aspartyl-phenylalanine) n according to any one of claims 25 and 27 is subjected to acidic or enzymatic hydrolysis. and in that the desired product is separated from the hydrolyzate.  32. A process according to claim 31, characterized in that the acidic hydrolysis agent is hydrochloric acid.  33. The process according to claim 31, characterized in that the enzymatic hydrolysis agent is carboxypeptidase, aminopeptidase or an aspecific endopeptidase.  34. L-Phenylalanine or L-aspartic acid when it has been produced by carrying out a process according to any one of claims 31 to 33.