NL8100437A - RECOMBINANT DNA TECHNIQUES; SYNTHETIC GEN; METHOD FOR THE PREPARATION THEREOF; PLASMIDE VECTOR; METHOD FOR THE PREPARATION THEREOF; CELL; METHOD FOR TRANSFORMING A CELL; METHOD FOR EXPIRING A PEPTIDE; SO PEPTIDE OBTAINED; (ASPARTYL-PHENYLALANINE) N; METHOD FOR PREPARING ASPARTYL-PHENYLALANINE METHYL ESTER; SO OBTAINED ASPARTYL-PHENYLALANINE METHYL ESTER; PROCESS FOR PREPARING L-PHENYLALANINE OR L-ASPARTINIC ACID. - Google Patents

Description

m t κ VO 1545

Recombinant DNA techniques; synthetic gene; process for the preparation thereof; plasmid vector; process for the preparation thereof; cell; method of transforming a cell; method of expressing a peptide; peptide thus obtained; (aspartyl-phenylalanine] n; process for preparing aspartyl-phenylalanine methyl ester; aspartyl-phenylalanine-methyl ester thus obtained; process for preparing L-phenylalanine or L-aspartic acid.

The invention relates to recombinant DNA techniques.

More particularly, the invention in its broadest aspect relates to a method for the preparation of oligo-peptides or polypeptides by recombinant DNA techniques, using a synthetic gene encoding a repeating polymer of the desired oligopeptide or polypeptide .

The invention also relates to such a synthetic gene and to its preparation.

The synthesis of peptides by chemical means is a laborious process, which becomes less and less efficient with increasing length of the peptide. On the other hand, in the preparation of peptides by microorganisms in a fermentation process, there is no variation in efficiency relationship to the length of the peptide product. However, short peptides are metabolically unstable in the organisms used in recombinant ÜNA processes. It is e.g. it is known that the peptide scmatcstatin, which consists of 14 amino acids, is not stable when directly in E_. coli is prepared. In that case, stability is obtained by coupling the peptide to a nor-2Q times E1. coli protein and then split this fusion product

Cltakura, k., Et al., Science 198, 105B-1063, 1977).

According to the present method, stability and increased yield were achieved using a synthetic 81 00 43 7 - 2 - β * gene containing a multiple sequence repeat encoding a desired small peptide. The product is therefore a large molecule, which is more metabolically stable in E_. coli is then the small peptide. The subsequent cleavage of the large molecule 5 yields the smaller peptide contained therein in a higher molar yield than would be obtained if the small peptide had been prepared directly using a monomer gene, even if the small peptide were metabolically stable .

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

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

•O

The complementary or non-coding strand of the synthetic gene contains an order determined by the order of the coding strand according to the accepted rules of nucleic acid base pairing.

In another embodiment, the invention relates to a method for the preparation of such a synthetic gene, which comprises the superstructure and ligation and, if desired, repair of a variety of suitable oligonucleotides, the structure of which is to achieve the repeating character. 25 is self-complementary overlapping ends at the endpoints of the group of oligonucleotides, which comprises the repeating unit of the gene after construction,

In a further embodiment, the invention relates to a plasmid vector comprising an insertion site introduced therein adjacent to a bacterial promoter downstream of a prokaryotic ribosome binding site, such a synthetic gene that the gene is under bacterial promoter control.

In yet another embodiment, the invention relates to a cell transformed by introducing such a plasmid vector into it.

In another embodiment, the invention relates to a method of expressing a peptide, wherein such a cell is cultured.

As indicated above, the strands of the synthetic gene are prepared from a series of short oligonucleotides which are themselves prepared by combining the required nucleotides by known methods in an order determined by the codons of the amino acids of the desired peptide and the under-1G are more detailed rules.

The oligonucleotides of the coding strand and the complementary strand of the gene form an overlapping set corresponding to the following general structure: ci 1 i .1 i 1 i, 1 i 1 i -1 5 'a ibicidieif i 3' "1 I- I 1- I - f - - - |

* I I t I I I

15 I_I_I I_ I _I C, 3 ~~ 2 i ΤΊ ~ 2! T! ~~ 3. 1 2 1 2 3 4 b i c i d i e i f i a

The structure shown here is composed of six oligonucleotides, three in each strand. However, the structure can be built up from any number of pairs of oligonucleotides from one pair upwards, provided that: 1 2 1 2 1

Cl] the paired regions, denoted a and a, b and b, c and 8100437 2 c .... nucleotide base sequences, which are complementary within the pair, but not to the sequences of any other pair, 12 12 12 C2] each of these regions a, a, b, b, c, c ... contain at least 3 four nucleotide bases; and 4 (3] the sum of the nucleotide bases in each strand, which ultimately comprises the polymer repeat length after construction, is divisible by three.

When a set of oligonucleotides of this character are mixed, the complementary regions of the oligonucleotide pairs hybridize to form high molecular weight structures, in which the sequences of the oligonucleotides Ca * - f ^ in the above picture] are repeated in tandem from a few turn up to 35 many cards, in different molecules. In the subsequent process, it is desirable to maximize the number of polymers having in the order a ^ at their 5 'endpoint.

This is accomplished by mixing the oligonucleotides a1 b ^, c * d ^, 1 1 2 2 2 2 ef, bc and the above image in equimolar 2 2 5 amounts and then adding the oligonucleotide fa in less than equimolar amount to add. Since the complements of a1 and f1 are present in submolar amounts, most polymers will end in a "* - and f *. In general, the 5 'terminal oligonucleotide of the complementary set is added to the polymerization mixture in a how much less than equimolar to the other oligonucleotides, all of which are present in equimolar amounts.

The "notches" in the chains, where adjacent oligo-nucleotides in the chain are adjacent, can be joined using the enzyme ÜNA compound CEC 6.5.1.1), a known procedure.

The molecules thus prepared will have single strand ends. These can be converted into the so-called "butt end" -20 form using the enzyme DNA polymerase I CEC 2.7.7.7) according to a known procedure.

The resulting molecules can be placed in competent microbe cells, in particular EE. coli are cloned using a desired vector. In particular, a plasmid vector selected from the so-called "pWT series", see, e.g., Tacon, et al., Molec. Gen.

Genet., 177, 427 (1980) are used, the vector being selected such that the synthetic gene, when inserted in the correct orientation, is read in the reading frame of the desired translation.

From the drawing, Fig. 1 shows a scheme for the preparation of a recombinant plasmid (pWT 121 (asp-phe) n) containing the repeating gene for (asp-phe) n in the correct DNA phasing to express a (asp-phe ^ 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 8100437-5 DNA polymerase I to form a blunt-ended molecule The Casp-phe) n coding polymer, shown at the bottom of the diagram, similarly repaired its initial protruding 5 'end to obtain another 5-blunt molecule. The combination of these two blunt-ended species with T4 DNA ligase gives the recombinant DNA, shown in the center of the diagram, with the correct DNA phasing for expressing (asp-phe] n.

The cloning of this gene, the choice of bacterial colonies, the detection of the polypeptide product of the gene expression and the extraction and purification of the polypeptide can all be performed according to known procedures.

This polypeptide can then be cleaved either enzymatically using specific enzymes or chemically, e.g. Democratic use of cyanogen bromide to cleave polypeptide chains in methionine residues.

As the skilled artisan will appreciate, the invention can be practiced in many ways. The invention can e.g. are used for the peptide hormones oyytocin, vasopressin and somatostatin, the enkephalins (opiate pentapeptides) and the appetite regulating peptides.

For illustrative purposes, one particular embodiment of the invention will now be described in more detail for the sweetener "Aspartame".

Aspartame is a low calorie artificial sweetener that can be described chemically as aspartyl phenylalanine methyl ester. Aspartame is usually prepared by a multi-stage chemical synthesis.

Because of its use as a sweetener, the total consumption of aspartame is potentially very high and therefore there is great interest in the development of preparations, where the material can be obtained appropriately and on a large scale.

It has been found that aspartame can be prepared by a potentially large-scale process based on recombinant DNA-tsch-81 0 0 43 7 Λ Λ - 6 -nics in which a synthetic gene encoding (aspartyl-phenylalanine) n is introduced into a clone vector with a controllable bacterial promoter located upstream of and substantially adjacent to the insertion site, and wherein the vector can be used to transform the bacterial cells from which the expressed polypeptide can be obtained and then split to form aspartyl phenylalanine and therefore obtain aspartame.

Thus, in a further embodiment, the invention relates to a synthetic gene for expressing the repeating polypeptide (aspartyl-phenylalanine), which comprises a double-stranded DNA molecule containing in the coding strand at least two nucleotide trimers encoding expressing aspartic acid (Asp) and alternatively at least two nucleotide trimers encoding the expression of phenylalanine (Phe) and in the other strand repeating and alternating nucleotide sequences complementary to the (Phe) and (Asp) coding sequences .

Therefore, the coding strand of the synthetic gene, ie the strand encoding the repeating polypeptide (Asp-Phe) n, can be represented as coding: 5 '- (Asp - Phe - Asp - Phe) n - 3' where n is a integer, e.g. 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 can be in any permutation with corresponding complementary sequences in the other strand are applied. The coding strand can e.g. have the following nucleotide sequence: 30 Phe Asp Phe Asp Phe Asp i-i ï-n-) <--> ï-1 5 "-T-T-T-C-G-A-C-T-T-C-G-A-T-T-T-C-G-A-C-T-T-3-A-C-A-C-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-G-A-

In this case, (T-T-C) is used as the sequence encoding for phenylalanine 35, and both codons (G-A-C) and (G-A-T) are alternatively 8100437-7 ft.

used for aspartic acid.

Those skilled in the art will understand the other possible permutations and combinations of nucleotide sequence for the coding and complementary strands.

The synthetic "Aspartame gene" according to the invention can be prepared by polymerizing a double strand fragment of DNA consisting of two dodecanucleotide strands, one with the sequence for expressing (Asp-Phe ^ * the coding strand and the other complementary.is, the complementary 10 strand.

Thus, in a further embodiment, the invention relates to such a method, providing a first dodecanucleotide sequence encoding aspartyl-phenyl-alanine by linking appropriate nucleotide monomers, providing a second dodecanucleotide sequence complementary to the first order, both dodecanucleotides are phosphorylated using polynucleotide kinase and the first and second consecutive are mixed together to form a double stranded DNA structure.

The dodecanucleotide strands can each be synthesized from the respective monamers which are known to be protected by blocking groups in a known manner and coupled according to a conventional phosphotriester methodology, see e.g. Hsiung et al. Nucleic Acids Research, 6, 1371 - 1385 C1979)], wherein the dodecamers thus obtained are deblocked and subsequently purified. After purification and phosphorylation with ΤΔ polynucleotide kinase (E.C. 2.7.1.78), the coding and complementary strands are mixed together, resulting in hydrogen bond double-strand structures. It has been found that using the dodecamer encoding CAsp-

Phe) 2 for the coding strand and a corresponding dodecanucleotide complementary strand, very stable double strand structures are obtained due to the overlapping of six bases of the strand, which can be shown in the given example as follows: 8100437 - a - 5 ' pT-TTCGACT: TrC-GA ^ 3 '3' GAAGCTAAAG-Tp 5 '

Linear polymerization of the above double-stranded structures is achieved by incubation with t4 DNA ligase (EC 6.5, KI) to yield long polymers of arbitrary length.

After separation of the non-ligated material, it is found that an area of polymers containing from 2 to 500 and more repeats of the base units is obtained.

The invention also relates to a method for expressing (aspartyl-phenylalanine), wherein such a synthetic gene is introduced into an insertion site of a plasmid vector that reads 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, competent microbe cells are transformed using the obtained plasmid vector, the transformed cells are cultured and the expressed peptide is collected. Moreover, the polymeric product is considered new,

The invention further relates to a method of preparing aspartame (the methyl ester of aspartyl-phenylalanine), wherein the above-mentioned synthetic gene is introduced into a plasmid clone vector, e.g. pWT 121, designed to read in the correct phase for expression of the inserted gene and with a bacterial promoter located upstream of and next to the insertion site. The plasmid vector can be used to find _E. coli HB 101 cells, collecting the expressed (Asp-Phe) n and subjecting to enzymatic cleavage using an amino acid with aromatic side chains, e.g. subtilisin (E.C. 3.4.4.16) chymotrypsin (E.C. 3.4.4.5) or proteinase K (E.C. 3.4.21.14) to give aspartyl phenylalanine which is methylated to prepare aspartame. The enzyme used can be used in soluble form or, preferably, immobilized on a solid support. Nethylation can be carried out by conventional chemical means or by an exchange reaction with the enzyme 8100437-9 in the presence of methanol or by direct enzymatic methanolysis of the polymer.

Given the triplet nature of the genetic code, it is clear that the gene can be read in any of the three phases and is therefore essential for a gene used as a piasmid vector to be translated into the correct reading frame.

It has been found that a plasmid vector which is preferably used in the present method is a vector selected from the above-mentioned so-called "pWT series".

Basically, the pWT series plasmids comprise a Hin dill (E.C. 3.1.23.21) restriction site adjacent to a cloned E.coli tryptophan promoter. Through Hin d III restriction and the insertion of Hin d III couplers, it is possible in the pWT series to construct 15 plasmids capable of translation in all three reading frames, which according to the description in the above referenced pWT 111, pWT 121 and pWT 131.

Transfer and translation of introduced DNA into the plasmids of the pWT series is under direct and strong control of tryptophan; the aperone is therefore suppressed in the presence of tryptophan while de-suppressed in the absence of tryptophan.

Plasmids of the pWT series also possess in the region immediately following the Hin d III site, the gene for tetracy-cline resistance, so that after insertion of the DNA, transfer and translation can be easily confirmed because when this has taken place , tetracycline resistance is maintained.

Therefore, in a preferred embodiment of the present method, the synthetic gene is introduced into the appropriate plasmid of the pWT series, i.e., the plasmid designated as "pWT 121". This plasmid is shown schematically in Figure 2 and has the following properties:

a measurement length of dB37 bc; a Hpa I (E.C. 3.1.23.233 place; a Hin d III place 206 bp from the Hpa I place; a Bam Hl 35 (E.C. 3.1.23.5) place 353 bp from the Hin ο III place; a Sal I

8100437 - 10 - CE.C. 3,1,23,37) site 275 bp from the Bam HI site; a Pst I CE.C. 3.1.23.31) pteats 2958 bp from the Sal I site and 1045 bp from the Hpa I site; the tetracycline resistance gene extends from the region of the Hpa I site beyond the Sal I site;

5 the gene for ampicillin resistance in the Pst I region

site and the cloned portion of the trp operon includes 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 of the invention was cloned 10 to pWT 121 in the following manner:

The plasmid pWT 121 was restrained with the enzyme Hin d III to obtain a linear molecule which was then treated with DNA polymerase and all four deoxyribonucleoside triphosphates to obtain fully base paired "blunt ends". The molecule blunt end was then treated with bacterial basic phosphatase CE.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 E1. coli DNA polymerase and all four deoxyribonucleoside triphosphates to obtain blunt ends and the "repaired" genes are separated from the enzyme and small molecules.

Blunt-ended vector DNA and blunt-ended gene material is mixed in a ratio of 1: 1 to 1: 2 and ligated together with high concentrations of DNA ligase to form the plasmid sequence "pWT 121 / (asp -phe) n ”The sizes of the cloned inserts, determined by restriction enzyme digestion, were found to range from 60 to 900 bp Cd (ie 5-75 repeats of the dodecanucleotide unit).

The pWT 121 / Casp-phe) plasmids described above are used to generate E_ in a known manner. transform coli cells, e.g. by exposure of calcium chloride treated cells to the pWT 121 / Casp-phe) plasmid. Transformed csl-35s, cultured in a known manner in a medium not containing 8100437-11-tryptophan and preferably containing initiator / 3-indolearicylic acid, resulted in expression of a protein consisting essentially of Casp-phenyl 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 multi-stage chemical synthesis. An intermediate used in this conventional synthesis is L-phenylalanine. The invention also relates to the preparation of L-phenylalanine which is useful in this conventional synthesis. When phenylalanine chemical synthesis is prepared, it is obtained as a racemate that must be cleaved before the L isomer can be obtained and this requires an additional and costly process step. When phenylalanine is obtained according to an embodiment of the invention, it is obtained directly as the L isomer.

Thus, the invention further relates to a method of preparing L-phenylalanine, wherein the [asp-phe] peptide is treated, which has been isolated from a culture of E. coli cells carrying plasmids in which the synthetic aspart-tame gene is incorporated, with an acidic or enzymatic hydrolysis agent, and the desired L-phenylalanine is separated from the hydrolyzate.

Naturally, this method also gives L-aspartic acid and the invention therefore also relates to such a method.

Preferred hydrolysis agents include hydrochloric acid or carbonypeptidase CE.C. 3.4.12.x], aminopeptidase CE.C. 3.4.11.x] or non-specific endcpeptidases.

The separation of the hydrolysis products can be effected by known methods.

The invention is further elucidated by means of the examples.

Example I Casp-pheJ ^

Synthesis of dodecanucleotides 35 2ε two dodecanucleotides TpCpGpApApApTpCpGpApA.pG and 8100437 - 12 - «

TpTpTpCpGpApCpTpTpCpGpA, were prepared according to a conventional triester methodology such as e.g. described by Hsiung, 1.c., and according to the reaction scheme shown in Figure 3, wherein N represents gisobu ^ j-.bz. The fully protected dodecanu-

5 cleotides were unblocked with 2% w / v benzenesulfonic acid, 0.1 M

tetraethylammonium fluoride in THF / pyridine / water CS: X s 1 volume ratio), followed by treatment with ammonia. The deblocked dodecanucleotides were purified by HPLC on "Partisil 10 SAX" (microscopic silica, derivatives of which were made with quaternary ammonium groups (Whatman)).

Polymerization of dodecanucleotides;

Each synthetic dodecanucleotide (2 µg) was phosphorylated in a 10 ydnn reaction containing 10 - 50 yCi of P-ATP, 50 mM tris-Cl pH 7.8, 5 mM magnesium chloride, 0.25 mM unlabeled 15 ATP, 10 mM mercaptoethanol and 3 units (1 unit is the amount causing the transfer of 1 nanomole of P-phosphate from r 22 X * P) -ATP to the 5% hydroxy endpoint of a polynucleotide in 30 minutes at 37 ° C under standard analysis conditions, see e.g. Richardson, Nucleic Acids Research, 2,815) of polynucleotide kinase (E.C. 2.7.1.78). After 60 minutes at 37 ° C, the two dodecanucleotides were mixed, unlabeled ATP was added to increase the ATP to 1 mM along with 0.1 units (1 unit is the amount that 100 monomoles of d (AT) 100Q will convert into an exonuclease III resistant form 25 in 30 minutes at 30 ° C under standard analysis conditions, see, e.g., Modrich and Lehman, J. Biol. Chem., 245, 3626 (1970) of T1 DNA ligase (EC 6.5.1.1) and reaction mixture incubated at 25 ° C for 24 hours ATP and non-ligated nucleotide monomers were removed by passing down through a 30 "Sephadex 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 dodecyl sulfate (SDS) The double stranded polymers were concentrated by ethanol precipitation The product was found to be a mixture after separation be of any length polymers ranging from 2 to over 81 0 0 43 7 - contain 13 - 5QQ repeats of the base units.

Clones of the synthetic gene.

Ca] Blunt end repair of the synthetic gene.

2 µg of the 3 'prepared as described above were incubated

5 polymer in a 40 µm mixture with 50 mM tris-Cl pH 7.8. 5 mM

magnesium chloride, 1 mM mercaptoethanol, 0.125 mM each. of the four deoxynucleotide triphosphates and 2 units Cl unit, the amount causing the uptake of 10 nanomoles of nucleotide in an acid is precipitable form in 30 minutes at 37 ° C under 10 standard analysis conditions using poly dCA-T] as template , see e.g. Richardson et al., J. Biol. Chem., 239, 222 C19643 of E. coli DNA polymerase I CE.C. 2.7.7.7]. The mixture was incubated at 10 ° C for 20 minutes and the mixture used without further purification.

Cb] Preparation of the clone vector pWT 121.

50 µg of pWT 121 DNA was digested with a two-fold excess of Hin d III CE.C. 3.1.23.21]. The mixture was extracted twice with an equal volume of phenol and precipitated with ethanol.

20 Repair with jï. coli DNA polymerase to obtain blunt ends was performed as above under Ca].

The blunt ended DNA was then treated with bacterial basic phosphatase CE.C. 3.1.3.1) to remove terminal phosphates and prevent it from recirculating 0ΝΑ during subsequent ligation. The DNA was incubated for 30 minutes at 37 ° C in the presence of a two-fold excess of enzyme in 10 mM tris-Cl pH 7.5, 0.1% SDS. The mixture was then exhaustively extracted with phenol, washed several times with chloroform and then precipitated with ethanol.

Cc) Stamp end-ligation.

Blunt-ended decanucleotide polymer from Ca) and blunt end-pWI 121 CNA from Cb) were mixed in a 1: 1 weight ratio and ligated at 15 ° C with 0.2 units of DNA-3 ligase in a 20 µm mixture containing 50 mM tris-Cl pH 7.3, 5 mM magne-81 0 0 43 7-14 sodium chloride, 1 mM ATP and 10 mM mercaptoethanol. After 24 hours, the pWT 121 / (asp-phe] n recombinant plasmids were ready to use to transform E. coli cells.

Transformation and gene expression: 5 E_. coli K12 HB 101 cells (genotype gal, lac, ara, pro, arg, strrj ree A, r ^ * M ^ 5 Boyer, HW and Roullard -Dussoix, D., J. Mol. Biol., 41, 459 - 472 ] were transformed according to the procedure of Katz et al (19731 J. Bacteriol, 114, 577 - 591, and loaded onto L-agar plates supplemented with ampicillin 10 (100 µg / cm 2).

Recombinant clones were purified by streaking to obtain single colonies. Clones thus isolated were assayed for colony hybridization on nitrocellulose filters (Grunstein and Hogness (1975), Proc. Nat. Acad. Sci. USA 72, 15 3961 - 3965] using a kinetic e-labeled synthetic dodecanucleotide as hybridization probe. Single colonies, positive by colony hybridization, were grown to a 3

AgQQ of 0.6 in 25 cm M9 medium (Miller (1972), Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, 3 20 biz.433] supplemented with ampicillin (100 µg / cm] and tryptophan 3 3 '( 40 µg / cm]. A 1 cm sample was taken from this repressed culture and labeled for 10 minutes with 5 yCi C-amino acids before being grooved for 10 minutes at 200 µm 20% w / w v "casamino acids" (Difco]. Cells were centrifuged ("pelleted"] (10,000 xg for 10 minutes], washed several times with phosphate buffered saline 3 containing 100 µg / cm gelatin to remove excess label "3 and the final pellet lysed in 50 ydm of a buffer (FSB] containing 10% v / v glycerol, 0.01% w / v bromophenol blue, 5% v / v / 3-30 mercaptoethanol, 3% w / v SOS and 65 mM tris-Cl contained pH 0 by heating for 2 minutes at SO 0 C. The rest of the initial 25 cm culture was pelleted (10,000 xg for 10 minutes] e n resuspended in an equal volume of M9 medium, supplemented with ampicillin (100 µg / cm] and 3-indole acrylic acid (5 µg / cm 2). After induction several times, samples of 8100437 3 - 15 - 1 cm were removed and labeled as described above. Portions of 3 final 5-10 µm lysates were separated on acrylamide gels C12.5% polyacrylamide + 0.1% SDS, see e.g. Laemmli,

Nature 227, B80 - S85 Q9703 3. The gels were dried and viewed radiographically to determine the positions of the labeled proteins. The comparison of the patterns of uninduced and induced cells could be identified synthesized proteins under the control of the tryptophan promoter.

Figure 4 shows an autoradiogram of a 12.5% acrylamide: 14 0.1% SDS gel on which C-labeled proteins were separated, which were synthesized in Ei. coli cells, which are recombinant pWT 121

Casp-phe] plasmids carried. Lane 1 shows proteins labeled 14-3 with C-amino acids in the presence of 40 µg / cm L-tryptophan, i.e. genes introduced into the Hin dII site will be repressed and no protein would be formed. Lane 2 shows proteins, 14 labeled with C-amino acids in the absence of L-tryptophan and 3 in the presence of 5 µg / cm3 / 3-indole acrylic acid, ie introduced genes will be fully induced and should be the protein encoded by the insert. express.

The protein that appears strongly in trace 2 was found to be a repeating polymer of Casp-phe3. 3 Traces 3 and 4 correspond to tracks 1 and 2, except that twice the amount of protein was used. The standard protein molecular weights indicated to the right of the picture are those of a standard protein mixture obtained from the Radiochemical

Center, Amersham, Buckinghamshire, England.

Isolation of labeled proteins from gels:

Certain protein bands were isolated from acrylamide gels by separating the protein lysate in a series of parallel spores. One of these spores was dissected from the gel with a dissecting knife and stained with Coomsssie Brilliant Blue R CGurr, Searle Diagnostics] while the rest of the gel was soaked in 15% v / v glycerol for 20 minutes and at -70 ° C saved. The colored gel disc was dried and autoradiographed for 24-43 hours to determine the position of the labeled bands. 8100437-16. This allowed excision of the relevant portion of the frozen gel. The gel disks were broken up by forcing them through a 1 cm disposable syringe without a needle and the labeled protein was eluted with 10 mM triethylammonium bicarbonate, pH 7.5, for 24 hours. Gel fragments were removed by centrifugation and the supernatant was dialyzed against 10 mM triethyl ammonium bicarbonate. In this way, yields of labeled protein bands of 50-75% were obtained. Enzyme digestions; Crude cell lysates or isolated bands of acrylamide gels were digested with proteolytic enzymes in solution at concentrations of 1-10 mg / cm, or with equivalent amounts of immobilized enzymes on a solid support, for periods of up to 72 hours. 10 mM triethylammonium bicarbonate was used for digestions in the pH range 7-8, while 10 mM glycine / sodium hydroxide buffers up to pH 10.5 were used.

Thin layer chromatography:

Enzyme digestions of labeled proteins were separated on Merck silica gel TLC plates with concentration zone C20 x 20 cm 3 using n-butanol acetic acid: water C8: 2: 2 volume ratio} or n-propanol: concentrated (0.8803 ammonia ( 7: 3 volume ratio 3 as solvent After development, the plates were dried and examined radiographically using Kodak "Kodirex" X-ray film to detect the labeled peptide products. Digestions with chymotrypsin (EC

3.4.4.53 or subtilisin (EC 3.4.4.163 immobilized on a solid support with subtilisin or proteinase K (EC3.4.21.143 in soluble form) all gave a mixture of products. In any case, there was a compound containing authentic aspartyl phenylalanine co-chromatographed. (Figure 5 of the drawing shows an autoradiogram of a silica thin layer chromatography plate on which the products are separated from an enzymatic digestion of (asp-phe3n protein. C-labeled (asp- phe3n protein, isolated from a 12.5% acrylamide: 0.1% SDS gel, was digested for 16 hours at 37 ° C with subtilisin, which had been immobilized on a solid support. The thin layer chromatography plate was developed with n-propanol: 0.880 ammonia (7: 3 v / v). The arrows point to the positions of markers of authentic (asp-phe) and (phe-asp). This compound, after recovery of the TLC-5 plate and hydrolysis in hydrochloric acid, gave only aspartic acid and phenyl alanine.

Hydrolysis of induced protein for the preparation of L-aspartic acid * and L-phenylalanine: 14

C-labeled (asp-phe) n protein isolated from an acrylamide gel, or a crude cell lysate, prepared by lysis of induced 14 C-labeled E_. coli cells carrying pWT 121 / Casp-phe) plasmids in a buffer containing 5% v / v / 3 mercaptoethanol, 3% w / v SDS and 75 mM trls-Cl pH 8 were used for hydrolysis. A 3 lysate, prepared from 1 cm of an induced cell culture, or the isolated induced protein of an equal volume of 3 culture, was enclosed in a tube with 1 cm 0 Π hydrochloric acid and heated at 110 ° C for 16 hours. After this time, the tube was opened and the contents removed and evaporated to dryness. Examination of the hydrolyzate by thin layer chromatography revealed the presence of C-labeled L-aspartic acid and L-phenylalanine, along with smaller amounts of other amino acids.

Example II

A polymer (asp-phe3 can be cleaved with the aid of an enzyme, such as chymotrypsin or subtilisin, which cuts through aromatic amino acids. On the other hand, a polymer (asp-phe-lysyl) can be obtained, which is obtained by trypsin or by an enzyme of similar specificity or by a combination of enzymes can be cleaved to form the dipeptide-asp-phe. For example, it is known that such cleavage occurs at paired base-30 amino acid residues in the process of cleaving hormone precursors to active species, e.g., the corticotropin / 3-lipotropin-nSH encephalin systema (Nakanishi, et al, Nature (137S), 278, 423-427J.

A gene for such a repeating tetrapeptide can be prepared by combining four chemically synthesized 8100437-18 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

(3) A.G.T.C.C.T.T.T.T.T.G.A

5 C4) A.A.T.C.T.T.T.C.T.T.A.A

forming a repeating structure: 8100437 - is - in * *! -

CO

C (- a. <L i- '- f— · -> · ν ^

a * «- <<CD

cn <«- * - '(0 <

CD

~. (- Γ c tn · a> <r-i | - η-

CD

01! - £ ^ u? H w H * 0) - (- JZ t- a. »Oj <

^ w <J

a “<| ω <C *

(C CD

.CD

t - (-

CD

cn a u ~ tSL * cn ~ '! - ~ Cm (->. c *.-i! -

<L

w «'. (- Γ c_j.

O * -------! - a cd, - · · - * ·

<J

f— ~ V) a. m <oi · C cc · <tr * g <. CD Ü LU (- C · i- ', ------- d <<CI .J ~ - (- m cn _i 8100437 - 20 -

This involves 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 Egg. coli. The only restriction enzyme recognition site within this polymeric gene is that for EcoRI '(A.G.A T.T.T).

Repair of the above polymeric gene using J5. coli DNA polymerase I gives a molecule that reads in the correct reading frame on blunt end ligation in the Hin d III site of the plasmid pWT 111, e.g.

The experimental procedure is as described in Example 1. The required polypeptide obtained on induction can be performed with e.g. trypsin are cleaved and the asp-phe dipeptide isolated.

Example III

Small amounts of the tripeptide Pyro-glu-his-gly have the ability to elicit an anorexic response in animals, i.e. it causes a reduction in food intake, and is therefore important in the control of appetite in humans. (D. Trustad et al., Acta Endocrinol, 8, 196-208 (1978)).

A polymer of the form (glu-his-gly-lys-lys) would use e.g. trypsin are cleaved to form the tripeptide glu-his-gly. Glutamic acid can be converted to pyroglutamic acid by the action of heat, see, e.g., U.S. Patent 2,528,267.

Such a repeating polymer is encoded by a repeating gene composed of four chemically synthesized oligonucleotides, two tetradecanucleotides and two hexadecanucleotides:

(1) A.G.G.A.A.C.A.C.G.G.T.A.A.G

30 (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 can be phosphorylated and ligated using the techniques described above to yield a structure: 8100437-21-cn in • "cj a I cn 05 · w

• Η <t— J

S

CJ CD

* · / - * · <C iH h— ^ <W f— ÖC ·

CD CJ

CD 0> <J H- H i <t-

W / I

* <H ^ cn *. w> <H- r-! «·

U CD

<H CD CJ

00 *

CD CJ

"F— CM <05 w · H <C H- JZ ·

a CD

- *

CD CJ

3 I — I <C i- 6C *

. CD CJ

• * <H- cn · ·> <t- r'è ^

CD S CJ

05 *> <H-cn i — 1. w. <* - ^ I · Γ f- <

CD CJ

00.

CD CJ

m m

* CJ CD

05 <- * • H <C I l—

J = w -C

CJ CD

* »,» Γ <* - 3 r-) <h-

Ofl · CD U "3-

9 I W

CD CJ

<in ch 8 1 0 0 43 7 - 22 -

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 these codons are in E_. coli acceptable. There are no restriction enzyme 5 sites in this gene. The experimental procedure is as described in Example 1. This gene reads in the correct reading frame when e.g. blunt-ended ligated into the Hin d III site of the plasmid pWT 111.

Example IV

The pentapeptide arg-lys-asp-val-tyr is an analog of the hormone thymopoietin, which has activity in inducing the differentiation of prosthymocytes into thymocytes (Goldstein et al., Science, (1979), 204, 1309 - 1310] It finds pharmaceutical application in the treatment of thymic ailments. A repeating gene encoding a polymer of this thymic hormone analog can be constructed from four chemically synthesized oligonucleotides, two tetradecanucleotides and two hexadecanucleotides:

(13 A.C.C.G.T.A.A.A.G.A.T.G.T.T.T.A 20 (23 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 can be phosphorylated and joined by ligation to form a structure: 25 8100437 - 23 - on <H '• ^ · (- r-i <«* -’ ·

cD CJ

• * cj cd f cj cd> <t Λ i “- <• · ·

CJ U CD

r-C · Ü · · «-» (0 (- <· <C 'S'> w

CD / CJ

w g Γ H- <a CD <i- <c

CD CJ

CD U, CD · <—> *>

> <OJ H

i-1 · '-'. <• · <H · bC ·

^ Ü CJ

ta

CJ CD

M m

CD U CD

£ <1 K.

rj · i- <cn

fl · <W

o h <>

, CD CJ

| - ^ a CD <f “03 · *

. CD U

• <t— cn - ^ ·> <. <- · i ~, i — 1 · *, <* “'(- <* *

in CD CJ

CD

U CD

CJ cd "<H- ϊΠ cn 8100437 - 24 -

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 CC.G.T and C.G.A), lysine (A.A.A and A.A.GJ and valine CG.T.T and G.T.C). There is a single restriction enzyme recognition site within the gene for the enzyme Acc I.

CG.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 introduced by blunt end ligation into the Hin d III site of the plasmid pWT 111, for example. The experimental procedure is as described in Example I.

Example V

The encephalins 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 importance. Two compounds are known, met-encephalin, which has the sequence (try-gly-gly-phe-met) and leu-encephalin, in which the terminal methionine has been replaced by leucine.

This example applies to met-encephalin, but a similar approach applies to leu-encephalin. For example, a polymer of (tyr-gly-gly-phe-met-lys-lys] can be cleaved by trypsin to form the required pentapeptide. Such a polymer is specified by a repeating gene constructed from six chemically synthesized oligonucleotides, two octadecanucleotides and four dodecanucleotides: 25 C1J GTATGGTGGATTTATGAA

C23 G -.- A.A.A.T.A.C.G.G.A.G.G.

C3] C.T.T.T.A.T.G.A.A.A.A.A.

C4) T.A.T.T.T.C.T.T.C.A.T.A.A.A.T.C.C.A.

CSJ A.T.A.A.A.G.C.C.T.C.C.G.

30, (6) C.C.A.T.A.C.T.T.T.T.T.C.

These oligonucleotides can be phosphorylated and joined by ligation in two ways, Either all six oliga nucleotides can be mixed and ligated to form the required polymer in one step, on the other hand, oligonucleotides 1, 2 and 4 and oligonucleotides 3, 5 and 6 are individually ligated 8100437 - 25 - into blocks, which can then be mixed and ligated to form the same polymer having the structure:} 8100437 - 26 - cn ui • \ uu <I- • (-n

<03 CJ

<t—

<H

"<H

cn · ·> <t- (—i · ·

<H

»* CO cj / /1 * ^ CD I — CH <ε. w l <"I- <

(D

x:! - <Q. * ·; Η. i

CJ M CO

t-i CO N rH f CJ CD

ÖO C · ^. co ε · u -

<· H

> · <, · «-! CO u Öfl

CO r— * CJ

JCN

CJ ^ CO

u · ς> <I- + j Λ H "^ I ·

<H

cn> i = C. (- hv

. <HH

«· M

co / cj cn-va ·> <> n · - I-r-c. ε. '

L <^ H

co CJ

P

QJI- <E · · - · «C I— _ · '-> I- <

CD

-Cf- <α - - I- H <--cr <c ^ o (-

> rH CJ

1-i CO W LU CJ

ÖO

"Co α <> ·

rH CO CJ

oo co u, “I— <c. . ^

> <H- CO

+ J. w CO u, cn cn 8100437 - 27 -

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 CT.A.T and T.A.C]; glycine (G.G.T, G.G.A and G.G.C] and lysine CA.A.A and A.A.G) 5 There are single recognition sites within the gene for the enzymes Mnl I (C.C.T.C), Mbo II CG.A.A.G. A) and EcoRI 'CG.G.A.T.T.T] as indicated above. These places repeat every 42 bases. The gene is designed to read in the correct reading frame when e.g. blunt-ended ligation is introduced into the Kin d III 10 site of the plasmid pWT 121.

8100437

Claims (48)

A synthetic gene, which contains codons in the coding strand for the amino acids of a desired peptide in tandem repeat. Synthetic gene according to claim 1, as essentially described here with particular reference to the examples and / or the drawing. The method of preparing a synthetic gene according to claim 1, which comprises the self-construction and ligation and, if desired, repair of a plurality of suitable oligonucleotides, wherein the construction to obtain the repeating character is processed by self-complementary overlapping ends at the endpoints of the group of oligonucleotides which, after construction, comprises the repeating unit of the gene. 4. Method according to claim 3, essentially as described here, in particular with reference to the examples and / or the drawing. Synthetic gene according to claim 1, prepared by a method according to claim 3 or 4. 6. Plasmid vector inserted therein at an insertion site, adjacent to a bacterial promoter and downstream of a procaryotic ribosome binding site, comprising a synthetic gene according to one or more of claims 1, 2 or 5, such that the gene is under bacterial supervisor check. Plasmid vector according to claim 6, essentially as described herein with particular reference to the examples and / or the drawing. A method for the preparation of a plasmid vector according to claim B, which comprises inserting a synthetic gene according to one or more of claims 1, 2 or 5 at the insertion site thereof. The method according to claim 8, essentially as described herein with particular reference to the examples and / or the drawing. 8100437 - 29 - The plasmid vector of claim 6 prepared by a method of claim 8 or 9. A cell transformed by the introduction of a plasmid vector according to any one of claims 6, 7 or 5. Cell according to claim 11, essentially as described herein with particular reference to the examples and / or the drawing. 13. A method of transforming a cell, wherein it is introduced into a plasmid vector according to one or more of claims 6, 7 or 10. A method according to claim 13, essentially as described herein with particular reference to the examples and / or the drawing. Cell transformed by a method according to claim 13 or 14. A method for the expression of a peptide, wherein a cell according to one or more of claims 11, 12 or 15 is cultured. The method of claim 16, essentially as described herein with particular reference to the examples and / or the drawing. 18, Peptide expressed by a method according to claim 16 or 17. 25 IS. Synthetic gene according to claim 1 for the expression of the repeating polypeptides Caspartyl-phenylalanine) n, wherein n represents an integer of at least 2, comprising a double-stranded DNA molecule comprising at least two nucleotide sequences encoding the coding strand. expression of aspartic acid, and alternatively comprising at least two nucleotide sequences encoding the expression of phenylalanine, and sequentially sequencing and alternating nucleotide sequences complementary to those cedar for aspartic acid and phenylalanine. Synthetic gene according to claim 19, having the structure 8100437 - 30 - 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' 21. A synthetic gene according to claim 19, essentially as described herein with particular reference to the examples and / or the drawing. 22. The method of claim 3 for the preparation of a synthetic gene according to claim 19, wherein a first dodecanucleotide sequence is encoded encoding aspartyl-phenyl-alanine, coupling the appropriate nucleotide monomers, a second dodecanucleotide provide sequence complementary to the first sequence, phosphorylate both dodecanucleotides using polynucleotide kinase and mix the first and second sequences together to form a double-stranded DNA structure. The method of claim 22, wherein the double-stranded structure is polymerized by incubating with T 1 DNA ligase. The method according to claim 22, essentially as described herein, with particular reference to the examples and / or the drawing. Synthetic gene according to claim 19, prepared by a method according to one or more of claims 22-24. The plasmid vector of claim 6, which is pWT 121, inserted therein into the Hin d III, instead comprising a synthetic gene according to any of claims 19-21 or 25. 27. The plasmid vector of claim 26, essentially as described herein with particular reference to the examples and / or the drawing. A method according to claim 8, for the preparation of a plasmid vector according to claim 26, wherein a synthetic gene 30 according to one or more of claims 19-21 or 25 is introduced into the Hin d III site of pWT 121. The method of claim 28, essentially as described herein with particular reference to the examples and / or the drawing. The plasmid vector of claim 26, prepared in a method of 8100437-31 according to claim 28 or 29. The cell of claim 11, which has a £. coli HB 101 is cell transformed by having a plasmid vector introduced therein according to any of claims 2S, 27 or 30. The cell of claim 31, essentially as described herein, with particular reference to the examples and / or the drawing. The method of claim 13 for transforming a cell containing an E_. coli HB 101 is oi, wherein a plasmid 1Q vector is introduced therein according to one or more of claims 2S, 27 or 30. The method of claim 33, essentially as described herein, with particular reference to the examples and / or the drawing. The cell of claim 31 transformed by a method of claim 33 or 34. A method according to claim 16 for the expression of a peptide, wherein a cell according to one or more of claims 31, 32 or 35 is cultured. 37. The method of claim 36, essentially as described herein with particular reference to the examples and / or the drawing. 38. Peptide expressed by a method according to claim 36 or 37. 39. [Aspartyl phenylalanine] n, wherein n represents an integer of at least 2. 40. A method according to claim 36 for the expression of Cas-partyl-phenylalanine], wherein a synthetic gene according to any one of claims 19-21 or 25 is introduced into an insertion site of a plasmid vector, which is in the reads correct phase for the inserted gene, where the insertion site is adjacent to a bacterial promoter and downstream of a prokaryotic rcbosome binding site, competents microbe cells are transformed using the resulting plasma vector, the transformed cells are cultured and the extracted - 8100437 - 32 - primed plasmid is collected. The method of claim 40, essentially as described herein with particular reference to the examples and / or the drawing. 42. (Aspartyl phenylalanine) n expressed by a method according to claim 40 or 41. A process for the preparation of aspartyl-phenylalanine methyl ester, wherein (aspartyl-phenylalanine) n according to claim 39 or 42 is subjected to enzymatic cleavage using an amino acid with aromatic side chains specific enzyme to obtain asparty-1-phenylalanine , and the product is methylated. The method of claim 43, wherein the enzyme used is chymotrypsin, subtilisin, or proteinase K. 45. The method of claim 43, essentially as described herein with particular reference to the examples and / or the drawing. 46. Asparty1-phenylalanine methyl ester obtained by a process according to one or more of claims 43-45. A process for the preparation of L-phenylalanine or L-aspartic acid, wherein (asparyl-phenylalanine) according to claim 39 or 42 is subjected to acidic or enzymatic hydrolysis and the desired product is separated from the hydrolyzate. 48. The method of claim 47, wherein the acidic hydrolyzant is hydrochloric acid. The method of claim 47, wherein the enzymatic hydrolysis agent is carboxypeptidase, aminopeptidase, or a nonspecific endopeptidase. 50. The method of claim 47, essentially as described herein, with particular reference to the examples and / or the drawing. 51. L-phenylalanine or L-aspartic acid, prepared by a method according to one or more of claims 47 - 50. 8 1 0 0 43 7