SE451595B - PLASMID VECTOR INCLUDING A SYNTHETIC GENE FOR EXPRESSION OF THE REPETITIVE POLYPEPTIDES (ASPARTYL-PHENYLALANIN) N, WHERE N IS AN INTEGRAL OF 2 AND E.COLI TRANSFORMED WITH THE VECTOR - Google Patents

Description

451 595 these at least two nucleotide sequences which encode the expression of phenylalanine, and in the second strand contain such repetitive and alternating nucleotide sequences which are complementary to the sequences encoding aspartic acid and phenylalanine, respectively.

The complementary or non-coding strand of the synthetic gene contains a sequence determined by the coding strand sequence in accordance with the established rules for base pair formation of nucleic acids.

The invention also relates to an E. coli cell which has been transformed by inserting such a plasmid vector therein.

The strands of the synthetic gene are prepared from a series of short oligonucleotides, which in turn are prepared by assembling the required nucleotides according to known methods in an order prescribed by the codons of the amino acids of the desired peptide and by the rules set forth below. .

The oligonucleotides of the coding strand and complementary strand of the gene form an overlapping structure, which is generally constructed as follows: 5 'al: Cl el 3' 3l This structure is composed of six oligonucleotides, three in each strand. But in fact the structure can be composed of any number of oligonucleotide pairs, thus of one or more (as many) pairs, provided that (1) those with ai and az, bï and bz, c] and c2. .. designated base pair regions contain nucleotide base sequences which are complementary within the pair in question but not complementary to the sequences of any other pair; (2) each of these regions ai, az, b1, bz, c1, AN * 451 595 c ..... contains at least four nucleotide base sequences; and (3) the sum of the nucleotide bases in each strand, which after assembly will comprise the repetitive sequence of the polymer, is divisible by 3.

When a group of oligonucleotides of this species are mixed together, the complementary regions of the oligonucleotide pairs hybridize to each other to form high molecular structures, in 1 in the above which the sequences of the oligonucleotides (a1 - f plotted scheme) are tandem repeated, and this in a variety number of times in different molecules, from a few times to many times. In the subsequent process, it is desirable to maximize the number of polymers in which the sequence a1 is present at the 5 'ends. This is achieved by mixing the above 1 bl 'cl 1 1 2 2 oligonucleotides a d1, e f, b c and d2 e2 in equimolar amounts and then adding the oligonucleotide f2 a 2 in an amount lower than the equimolar one. Since the complements to al and fï are present in submolar 1 and f1 at the ends. Generally, the amounts of the complementary set, most polymers will have a 5 'terminal oligonucleotide added to the polymerization mixture in an amount less than the equimolar relative to the other oligonucleotides, all of which are present in equimolar amounts.

The gaps (nodules) in the chains, namely at the joints between adjacent oligonucleotides in the chain, can be filled by splicing together with the enzyme DNA ligase (EC.6.5.1.1), which is a known method.

The molecules thus prepared have single-stranded ends.

These can be converted to ends with parallel strands using the enzyme DNA polymerase I (EC.2.7.7.7) according to a known method.

The molecules thus obtained can be cloned into cells of E. coli using a desired vector. In particular, one can use a plasmid vector selected from the so-called "pWT series", see for example Tacon et al, Molec Gen Genet 177, 427 (1980); the vector is selected so that the synthetic gene when inserted in the correct orientation 451 595 Fig. 1 shows a scheme for the preparation of a recombinant plasmid (pWT 121 (Asp-Phe) n), which contains the repetitive gene for (Asp-Phe) n the correct DNA phase for the expression of an (Asp-Phe) n protein. The DNA of the plasmid, shown at the top of Figure 1, is digested with the restriction enzyme Hind III and then repaired with E. coli DNA polymerase I to form a molecule with In the (Asp-Phe) n coding polymer shown at the bottom of Fig. 1, the initially single-stranded 5 'end is repaired in a similar manner, so that here too a molecule is obtained where the end has parallel strand ends. When these two types of molecules with adjacent strand ends are linked together by T4 DNA ligase, holding the recombinant DNA shown in the middle of Fig. 1, which has the correct DNA reading frame phase for expression of (Asp-Phe) n.

Cloning of this gene, selection of bacterial colonies, detection of the polypeptide product obtained by the expression of the gene and extraction and purification of the polypeptide are procedures, all of which can be performed according to known methods.

This polypeptide can then be cleaved, either enzymatically using specific enzymes or also chemically, for example by using cyanogen bromide to cleave polypeptide chains at methionine residues.

For illustrative purposes, a particular embodiment of the invention will be described in greater detail below, more particularly the application of the sweetener "Aspartame".

Aspartame is a synthetic low calorie sweetener, which can be chemically said to be aspartyl-phenylalanine methyl ester. Aspartame is currently manufactured in accordance with a multi-step chemical synthesis process.

Because Aspartame is used as a sweetener, the total consumption of Aspartame can be potentially very large.

Therefore, there is a great interest in developing methods by means of which the material in question can be obtained in a convenient manner and on a large scale.

It has been found that Aspartame can be prepared by a potentially large scale method based on recombinant DNA technology, in that a synthetic gene encoding (aspartyl-phenylalanine) n is inserted into a cloning vector having an un] 451 595 controllable bacterial promoter upstream of and substantially adjacent to the site of insertion, and the vector can be used to transform bacterial cells from which the polypeptide formed can be obtained, whereupon the polypeptide can be cleaved to obtain aspartyl-phenylalanine and thus also aspartame.

The synthetic gene for expression of the repetitive polypeptide (aspartyl-phenylalanine) n comprises a double-stranded DNA molecule, which in its coding strand contains at least two nucleotide trimers which encode the expression of aspartic acid (Asp) and alternately with said trimers. at least two nucleotide trimers encoding the expression of phenylalanine (Phe), the second strand containing repeating and alternating nucleotide sequences complementary to the (Phe) and (Asp) coding sequences.

The coding strand of the synthetic gene, i.e. the strand encoding the repetitive polypeptide (Asp-Phe) n, can thus be indicated as coding as follows: 5 '- (Asp-Phe-Asp-Phe) n - 3' where n represents a integers of, for example, up to several hundred.

There are two possible codons for Phe, namely (TTT) and (TTC), and two possible codons for Asp, namely (GAT) and (GAC), and these alternative variants can be used completely indiscriminately instead of each other, with against corresponding complementary sequences in the second strand. For example, the coding strand may be a nucleotide sequence Phe Asp Phe Asp Phe Asp y 1 | now, fffl 5 '-T ~ TTCGACT ~ TCGATTTCGACTT- 3' with a complementary strand whose sequence is 3 '-AAAGcTGAAGcTAAAGcTGAA- 5' In this case (TTC) was used as the coding sequence of the phenylalanine and both codons (GAC) and (GAT) were used ) alternating for aspartic acid. It will be clear to those skilled in the art what other permutations and combinations may be considered for the coding nucleotide sequences and what structures the complementary strands will then exhibit.

The synthetic "Aspartame gene" of the present invention can be prepared by polymerizing a double-stranded fragment of DNA, consisting of two dodecanucleotide strands, one with the sequence for expression of (Asp-Phe) 2 = the coding strand, and the other, i.e. the complementary string, exhibiting complementary bases to the bases of the coding string.

According to the present invention, there can be provided a first dodecanucleotide coding sequence for aspartyl-phenylalanine by linking appropriate nucleotide monomers, providing a second dodecanucleotide sequence complementary to the first sequence, phosphorylating both dodecanucleotides and using and the other sequences to form a two-stranded DNA structure. each of the dodecanucleotide strands can be synthesized from respective monomers, which are protected by blocking groups in known manner and linked by conventional phosphotriester methodology [see, e.g., Hsiung et al, Nucleic Acids Research 6, 1371-1385 (1979)]; from the dodecamers thus obtained, the blocking groups are removed, whereupon the dodecamers are purified. After purification and phosphorylation with T4 polynucleotide kinase (EC.2.7.1.78), the coding and complementary strands are mixed. As a result, double-stranded products are formed due to hydrogen bonds. It has been found that by using the dodecamer coding for (Asp-Phe) 2 as coding strand and a corresponding dodecane nucleotide as complementary strand, very stable double-stranded structures are obtained, which are formed as a result of the 6-base overlap of the strands.

For the above examples, this can be written 5 'pT-TTC ~ GACT ~ TCGA 3' 3 'GAAGCTAAAGC ~ Tp 5' 451 595 Linear polymerization of the above double-stranded structures 4 DNA ligases (EC.6.5.1.1) to form long polymers ( the lengths are random). After separation of non-ligated material, a collection of polymers containing from 2 to 500 and even more repeats of the basic units is obtained by incubation with T.

Expression of (aspartyl-phenylalanine) n is accomplished by inserting such a synthetic gene into an insertion site of a reading frame with a plasmid vector in the correct phase of the inserted gene, the insertion site being adjacent to a bacterial promoter and downstream of a prokaryotic tribosome binding site. using the plasmid vector thus obtained, competent microbial cells are transformed, the transformed cells are cultured and the peptide formed is taken care of.

In the preparation of Aspartame (the methyl ester of aspartyl-phenylalanine), the above-mentioned synthetic gene is inserted into a plasmid vector for cloning, for example pWT 121, which is constructed with a reading frame in the correct phase for expression of the inserted gene and which has a bacterial promoter upstream of and next to the deposit point. The plasmid vector can be used to transform E. coli HB 101 cells, and the formed (Asp-Phe) n can be recovered and subjected to enzymatic cleavage using an enzyme specific for amino acids with aromatic side chains, such as subtilisin (EC .3.4.4.16), chymotrypsin (EC.3.4.4.5) or proteinase K (EC.3.4.21.14), to obtain aspartyl-phenylalanine which is methylated to form Aspartame. The enzyme used may be in a soluble form or may - preferably - be immobilized on a solid support. The methylation can be carried out either 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.

Since the genetic code is based on triplets, the gene can in principle be read in three different phases. It is therefore important for the gene that it is inserted into such a plasmid vector that provides a translation in the correct reading frame. It has been found that a preferred plasmid vector for use in the present method is a vector of the above-mentioned "pWT series".

A distinctive feature of plasmids belonging to the pWT series is that they have a Hind III (EC.3.1.23.21) restriction site adjacent to a cloned E coli tryptophan promoter. By restricting Hind III and inserting Hind III connection links, it is possible in the pWT series to construct plasmids that can translate into all three reading frames. These plasmids are pWT 111, pWT 121 and pWT 131 as described in the above reference.

Transcription and translation of inserted DNA into plasmids belonging to the pWT series takes place under direct and strong tryptophan control; the operon is thus suppressed in the presence of tryptophan, while the suppression of the tofa is lifted. in the absence of tryp- In the region immediately after Hin d III site, the plasmids in the pWT series also show gene through tetracycline resistance; therefore, after DNA insertion, transcription and translation can be easily confirmed: If transcription and translation have taken place, tetracycline resistance is maintained.

According to a preferred embodiment of the method, therefore, the synthetic gene must be inserted into the appropriate plasmid from the pWT- series, i.e. plasmid "pWT 121". This plasmid is shown schematically in Fig. 2. It has the following characteristics: Molecular length U837 bp; an Hpa I (E.C. 3.l.23.23) site; a Hin d III place 206 bp from Hpa Instead; a Bam HI (E.C. 3.l.23.6) site 355 bp from the Hin d III site; a Sal I (E.C. 3.l.25.57) site 275 bp from the Bam HI site; a Pst I (E.C. 5.1. 23.31) site 2958 bp from Sal I instead and lOU5 bp from Hpa I instead; the gene for tetracine resistance extends from the Hpa I site region to a site beyond Sal I site; the gene for ampicillin resistance in the region of the Pst I site and the cloned part of the trp operon comprises the region between the promoter and the first part of the E gene between the Hpa I and Hin d III sites.

The synthetic aspartame gene of the present invention was cloned into pWT 121 as follows: The plasmid pWT 121 was restricted with the enzyme Hin d III to form a linear molecule, which was then treated with DNA polymerase and all four deoxyribonucleoside triphosphates. to form completely base-paired end portions, with the string ends lying "smooth". This molecule with adjacent strand ends was then treated with bacterial alkaline phosphatase (E.C. 3.1.3.1) to remove the 5 'phosphates. Following this treatment, the plasmid is 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 form adjacent strand ends, and the "repaired" genes are separated. from the enzyme and small molecules.

Vector DNA with adjacent strand ends and gene material with adjacent strand ends are mixed in a ratio of 1: 1 to 1: 2 and ligated together with high concentrations of T4 DNA ligase to form the plasmid series "pWT 121 / (Asp- - Phe) n ". The sizes of the cloned inserts were determined by restriction enzyme digestion and were found to be 60-900 bp (ie 5-75 repeats of the dodecanucleotide unit).

The pWT 121 / (Asp-Phe) n plasmids prepared as described above were used to transform E. coli cells in a known manner, for example by exposing calcium chloride-treated cells to the pWT 121 / (Asp-Phe) n plasmid. When transformed cells were grown in a known manner in tryptophan-free medium, which preferably contained β-indolacrylic acid, the cells formed a protein consisting essentially of (Asp-Phe) n, which after enzymatic cleavage and methylation gave the desired aspartyl-phenylalanine methyl ester. Aspartame.

As mentioned above, the product Aspartame is conventionally obtained by a chemical synthesis which is carried out in several steps. An intermediate used in this conventional synthesis is L-phenylalanine. If phenylalanine is prepared by chemical synthesis methods, this compound is obtained in the form of a racemate, which must be subjected to dissolution in its isomers so that the L-isomer can be obtained. This requires an additional, expensive process step. In contrast, when phenylalanine is prepared according to an embodiment of the present invention, it is obtained directly in the form of the L-isomer. The (Asp-Phe) n-peptide, isolated from a culture of E. coli cells containing plasmids with the synthetic Aspartame gene, is hereby treated with an acidic or enzymatic hydrolyzing agent, whereupon the desired L-phenylalanine is separated from the hydrolyzate. In this process, of course, L-aspartic acid is also obtained.

Preferred hydrolyzing agents include hydrochloric acid or kerbexipeptiaae (Ec.3.4.12.x), aminepeptiaae (Ec.3.4.11.- x) or non-specific endopeptidases.

The separation of the hydrolysis products can be accomplished according to known methods.

The invention is further illustrated in the following examples.

Example Gel 1 (Asp-Phe) n Synthesis of Dodecanucleotides The two dodecanucleotides TpCpGpApApApTpCpGpApApG and TpTpTpCpGp ApCpTpTpCpGpA were synthesized by conventional triester methods as described in, for example, Hsi. cit. and in accordance with the reaction scheme shown in Fig. 3. N denotes Fig. 3. From the completely protected dodecanucleic acid, T, cbz or Abz leotides, the blocking groups were removed with 2% (w / v) benzenesulfonic acid, 0.1 M tetraethylammonium fluoride in THF / pyridine / water (volume ratio 8: 1: l), with subsequent treatment with ammonia. The dodecanucleotides freed from the blocking groups were purified by HPLC on Partisil Sax (microparticulate silica derivatized with quaternary ammonium groups (whatmanb).

Polymerization of dodecanucleotides Each synthetic dodecanucleotide (2 pg) was phosphorylated in a 149 reaction medium containing 10-50; 1Ci 32 P-ATP, 50 mM Tris-Cl, pH 7.8, 5 mM magnesium chloride, 0.25 mM unlabeled. ATP, 10 mM mercaptoethanol and 3 units of T4 polynucleotide kinase (EC.2.7.1.78). (1 unit is the amount which, under standard conditions, causes the transfer of 1 nanomole of the 32 P feed from γ (32 P) -ATP to the 5 'hyarexi end of a polynucleotide within 30 minutes at 37 ° C, see for example Richardson, -Nucleic Acids Research, 2, 815). After 60 minutes at 37 ° C, the two dodecanucleotides were mixed, unlabeled ATP was added to increase the ATP content to 1 mM, together with 0.1 unit (as will soon be defined) of T4 DNA ligase ( EC.6.5.1.1), and the reaction mixture was incubated for 24 hours at 25 ° C. (1 unit in this context is the amount which converts 100 monomol d (AT) 1,000 to an exonuclease III resistant form within 30 minutes at 30 ° C under standard conditions, see for example Modrich and Lehman, J Biol Chem, 245, 3626 (1970).) ATP and non-ligated nucleotide monomers were removed by downward passage on a column of "Sephadex G-50" (20 cm x 0.8 cm) in 50 mM sodium chloride, 10 mM Tris-Cl, pH 7 .5, 0.2% (w / v) sodium dodecyl sulfate (SDS).

"Sephadex" is a trademark; "Sephadex G-50" is a superfine particulate modified dextran polymer with crosslinks, Pharmacia. The double-stranded polymers were concentrated by precipitation with ethanol. After separation, the product was found to be a mixture of polymers of random lengths, containing from 2 to more than 500 repeats of the base units.

Cloning of the synthetic gene (a) Repair of the synthetic gene to form parallel strand ends Zlug polymer, prepared as described above, was incubated in UO, ul mixture with 50 mM Tris-Cl, pH 7.8, 5 mM magnesium chloride, 1 mM mercaptoethanol, 0.125 mM of each of the four deoxynucleoside triphosphates and 2 units of E. coli DNA polymerase I (EC 2.7.7.7). In this context, by 1 unit is meant the amount which brings about the incorporation of 10 nanomoles of nucleotide in an acid precipitable form inon = 30 minutes at 3700 under standard conditions and using poly d (A-T) as a template; see e.g. Richardson, et al. J. Biol. Chem., Ëâg, 222, (l96 fl).

The mixture was incubated for 20 minutes at 1000, and the mixture thus obtained was used without further purification. (b) Preparation of the cloning vector pWT 12l 50 / ug pWT 121 DNA was digested with a double excess of Hin d Ill (E.C. 3.l.23.2l). The mixture was extracted twice with the same volume of phenol and subjected to precipitation with ethanol.

Repair with E. coli DNA polymerase to form adjacent strand ends was performed in the manner described in (a) above.

The resulting DNA, which had adjacent strand ends, was treated with bacterial alkaline phosphatase (EC 3.1.5.1) to remove the end-of-life phosphates and to prevent aüzdaum mwxates would change to ring form. during the 451 595 12 subsequent ligation. The DNA thus treated was incubated for 30 minutes at 3700 in the presence of a 2-fold excess of enzyme in 10 NM TT 1 S'Cl, PH 7.5, 0.1, 1% SDS. The mixture was then extracted completely with phenol, washed several times with chloroform and then precipitated with ethanol. (0) Mutal end-to-end ligation In a 1zl weight ratio, dodecanucleotide polymer with equal strand ends, obtained according to (a) above, and pWT 121 DNA with equal strand ends, obtained according to (b) above, were mixed and ligated at 1500 with 0.2 units of Tu DNA ligase in a 20 ul mixture containing 50 mM Tris-Cl, pH 7.8, 5 mM magnesium chloride, -1 mM ATP and 10 mM mercaptoethanol. After 2 h, the pWT 121 / (ASP-Phenfrecombinant plasmids were ready for use in transforming E. coli cells.

Transformation and gene expression E coli K12 HB 101 cells (genotype gal_, lac_, ara-, pro-, arg-, straw, recA_, rk_, Mk_; Boyer, HW and Roullard- -Dussoix, D, J Mol Biol, 41, 459 -472) was transformed according to the procedure described by Katz et al (1973), J Bacteriol, 114, 577-591, and deposited on L-agar plates supplemented with 100 pg / ml ampicillin.

The recombinant clones were purified by plating to obtain individual colonies. Thus isolated clones were tested by colony hybridization on nitrocellulose filters, (Grunstein and Hogness (1975), Proc Nat Acad Sci, USA 72, 3961-3965) using a kinase-labeled synthetic dodecanucleotide as hybridizing agent. Individual clones, which were found to be positive during colony hybridization, were grown to an A600 value of 0.6 in 25 ml of medium M9 (Miller, 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, p. 433), which medium was added with ampicillin (100 ug / ml) and tryptophan (40 ng / ml). A 1 ml sample was taken from this suppressed culture and labeled by 10 minutes treatment with 5 μCi 14C amino acids before being treated for 10 minutes with 200 μl 20% (w / v) "casamino acids" (Dífco).

The cells were centrifuged (10,000 xg, 10 min), washed several times with phosphate buffered saline, containing 100 μg / ml gelatin, to remove excess labeling agents in C 451 595 13] 4C compounds, and the finally obtained centrifuged grout residue was subjected to lysis in 50 μl of a buffer (FSB) containing 10% (v / v) glycerol, 0.01% (w / v) bromophenol blue, 5% (v / v) B-mercaptoethanol, 3% (w / v) SDS and 65 mM Tris-Cl, pH 8; the lysis was accomplished by heating to 90 ° C for 2 minutes. The residue of the original 25 ml culture was centrifuged (10,000 x g, 10 min) and resuspended in the same volume of medium M9, supplemented with 100 pg / ml ampicillin and 5 pg / ml B-indolacrylic acid. After different induction times, 1 ml of sample was taken and isotopically labeled as described above. 5-10 μl 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 determine By comparing the patterns from uninduced cells and induced cells, it was possible to identify the proteins synthesized under the control of the tryptophan promoter.

Hig. H shows an autoradiogram of a gel of 12.5% acrylamide; 0.1% SDS. This shows separated luC-labeled proteins, synthetic Sefade in E-001 cells, which contain recombinant plasmids PWT 121 (ASP-Phe) n. Lane 1 shows proteins labeled with luC-ami- HOSYPOP in the presence of 40 .mu.g / ml L-tryptophan, i.e. all possible genes inserted at the Hin d III site are in this case sub- Üfyßktä, so that in% ñt protein should be produced. Lane 2 shows proteins labeled with C-amino acids in the absence of L-tryptophan and in näPVaPO of 5μg / ml B-indolacrylic acid, i.e. in this case, the inserted genes will be fully induced, so that they should form the protein indicated by the insert code. [The protein, which appears strongly in lane 2, has been shown to be a repetitive polymer aV (ASD-Phe). ] Lane 3 resp. lane 4 corresponds to lane l resp. lane 2, except that twice the amount of protein has been used. The standard protein molecular weights listed above are those of a standard mixture of proteins obtained from the Radiochemical Center, Amersham, Buckinghamshire, England.

Isolation of isotope-labeled proteins from gels Specific bands of proteins were isolated from acrylamide gels by subjecting the protein lysate to separation in a series of parallel paths. One of these lanes was cut from the gel with a scalpel AND stained with Coomassie Brillant Blue R (Gurr, Searle Diagnostics), while the rest of the gel was soaked for 15 minutes in 15% (v / v) glycerol and stored at -7000. The stained gel disc was dried and autoradiographed for a period of 24-H8 hours to define the position of the isotope-labeled bands. In this way, it will be possible to cut out the interesting part of the frozen gel.

The gel slices were broken by pressing through a 1 ml disposable syringe without a needle, and the isotope-labeled protein was eluted for 24 hours with 10 mm triethylammonium bicarbonate, pH 7.5.

Gel fragments were removed by centrifugation, and the supernatant was dialyzed against 10 mM triethylammonium bicarbonate. 50-75% "yields" of isotope-labeled protein bands were obtained in this way.

Reactions with enzymes' Unclean cell dimuzell isolated bands from acrylamide gels were reacted either with proteolytic enzymes in solution, at concentrations of 1-10 mg / ml, or with equivalent amounts of enzyme immobilized on a solid support; the sales were carried out for periods of up to 72 hours. 10 mM triethylammonium bicarbonate was used for reactions in the pH range 7-8, while 10 mM glycine / sodium hydroxide buffer tar was used at up to pH 10.5.

Thin layer chromatography Enzyme conversion products of isotope-labeled proteins were separated on TLC-designated Merck silica gel plates with a concentration zone (20 x 20 cm), using either an 8: 2: 2 (v / v) mixture of n ~ _but & n0l: acetic acid: water or a 7: 3 ( volume) mixture of n-propanol-concentrated (0.880) ammonia as solvent. After development, the plates were dried and autoradiographed using Kodak "Kodirex" X-ray film to detect the isotope-labeled peptide products. The reaction with chymotrypsin (EC 3.ü.U.5) or subtilisin (EC 3.P.ä.l6) immobilized on a solid support, or with subtilisin or proteinase K (EC 3.4.2l.lH) in soluble form always gave a mixture of products. Among these was in each case a compound which on chromatography moved together with authentic aspartyl-phenylalanine. (Fig. 5 shows an autoradiogram of a TLC silica plate on which the products of enzymatic reaction of (Aqoime) n-protein are separated. With luC-labeled (A fl drhe) n-protein, isolated from a 12.5% euylamide: 0.1% SDS gel was reacted for 16 hours at 3700 with subtilisin immobilized on a solid support. The TLC plate was developed with n-propanol: 0.880 ammonia (7: 3 v / v). The arrows show the positions for use as markers, authentic (Amrrhe) and (Phe4mp).) When the above compound was recovered from the TLC plate and hydrolyzed in hydrochloric acid, it gave only aspartic acid and phenylalanine.

Hydrolysis of induced protein to produce L-aspartic acid and L-phenylalanine lnC-labeled (Asp-Phe) n-protein isolated from an acrylamide gel, or an impure cell lysate, prepared by lysing induced luC-labeled E. coli cells, containing pWT 121 / (Asp-Phe) n plasmids, in a buffer containing 5% (v / v) β-mercaptoethanol, 3% (w / v) SDS and 65 mM Tris-Cl, pH 8. A lysate prepared from 1 ml of induced cell culture, or the isolated induced protein from a similar culture volume was sealed in a tube with 1 ml of 6 M hydrochloric acid and heated for 16 hours to 10 ° C. After this time, the tube was opened and the contents were removed. For the hydrolysis, it was used out and evaporated to dryness. Then the hydrolyzate under- _. . 1 ~ was searched by thin layer chromatography, this showed that 4C-mark L-phaspartic acid and L-phenylalanine were present, as well as smaller amounts of other amino acids.

Example 2 A polymer (Asp-Phe) n can be cleaved by an enzyme such as chymotrypsin or subtilisin, which cuts into aromatic amino acids.

Alternatively, a polymer (Asp-Phe-Lys-Lys) n may be formed, which may be cleaved by trypsin or by an enzyme of similar specificity or by a combination of enzymes to form the dipeptide asp-phe. It is known, for example, that such cleavage at paired basic amino acid residues takes place in connection with the process in which hormone precursors are cleaved into active substances, e.g. corticotropin - B-lipotropin-MSH-enkephalin ”system (Nakanishi, et al., Nature, (1979), g1§, u25-h27).

A gene for such a repetitive tetrapeptide can be prepared by assembling four chemically synthesized dodecanucleotides: (1) AAGATTTcAAAA (2) AcGAcTTTAAGA (3) AGTCCTTTTTGA (4) AATCTTTCTTAA 451. 595 16 to form a repulsive structure: lys asp phe lys lys asp phe -raf- fi f- “Wf- 'fi r-àr-' fi f-xr- * fi f- fl 5 'AAGATTTCAAAAAGGActv.fr.TAZ \ .GAAAGATTTT' IL» nm 'n I nn ~ ( 2) (1) EcoRI '3' TTCTAAAGTTTTTCCTGAAAT.TCTTTCTAAAA5 'U) / l-è m <4) <3 »<4) (a) This involves the use of two codons for each of the amino acids, namely GAT and GAC for aspen; T.T.T and T.T.C for phe; A.A.A and A.A.G for lys. All of these codons are acceptable in E. coli. The only recognition site for a restriction enzyme in this polymeric gene is the site of EcoRI '(A.G.A T.T.T).

When repairing the above-mentioned polymeric gene using E. coli DNA polymerase I, a molecule is obtained which gives a reading in the correct reading frame phase when ligating dumb end to end into Hin d III instead of e.g. plasmid pwT 111.

The process itself is carried out in the same manner as described in Example 1. The required polypeptide obtained by induction can be cleaved using, for example, trypsin, and the Asp-Phe dipeptide can be isolated.

V)

Claims (4)

451 595 17 P a t e n t k r a v A plasmid vector, characterized in that a synthetic gene is inserted therein at an insertion site adjacent to a bacterial promoter and downstream of a prokaryotic tribosome binding site such that the gene is under the control of the bacterial promoter, said gene is a gene for expression of the repetitive polypeptides (aspartyl-phenylalanine) n where n is an integer of at least 2, comprising a double-stranded DNA molecule, which in the coding strand contains at least two nucleotide sequences which encode expression of aspartic acid and alternating with these at least two nucleotide sequences which encode the expression of phenylalanine, and in the second strand contains such repetitive and alternating nucleotide sequences which are complementary to the sequences encoding aspartic acid and phenylalanine, respectively. Plasmid vector according to claim 1, characterized in that the gene has the following structure: Vi 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' where n is at least 2 integers. Plasmid vector according to claim 1 or 2, characterized in that it is pWT 121, and that the gene is inserted at the Hin d III site. E. coli transformed with a plasmid vector according to any one of claims 1-3.