IE851973L - Polypeptide having hirudin activity - Google Patents

Abstract

Hirudin can be obtained using a specific DNA sequence in a genetic engineering process. The gene is advantageously synthesised in the form of several fragments which are enzymatically ligated to give two larger part-sequences which are incorporated into hybrid plasmids and amplified in host organisms. The part-sequences are reisolated and then combined to form the complete gene which is incorporated into a hybrid plasmid and the latter is expressed in a host organism. <IMAGE>

Description

58485 Hirudin is a polypeptide which is obtained from Hirudo medicinalis, has a specific antithrombin activity and is used as an anticoagulant.

It has now been found that polypeptides of the general formula I (X)m-A-B-C-Ty r-D-Asp-Cys-E-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu- -Cys-Glu-Gly-Ser-Asn-Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile- -Leu-Gly-Ser-Asp-F-G-Lys-Asn-Gln-Cys-Val-Thr-Gly-Glu- -G ly-Thr-Pro-Lys-Pro-Gin-Ser-His-Asn-A sp-Gly-A sp-Phe-G lu- -H-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-(Z) -OH n in which (I) m = 0 - 50/ n = 0 - 100, and X represents identical or different residues of genetically codable amino acids, A denotes lie or a direct bond/ B denotes lie, Thr or a direct bond, C denotes Thr, Val, lie, Leu or Phe, D denotes Thr or a direct bond, E denotes Thr or lie, F denotes Gly or a direct bond, G denotes Glu or a direct bond, H denotes Glu or Pro, and Z represents identical or different residues of genetically codable amino acids, and in which, where appropriate, the 6 Cys residues are linked pairwise via disulfide bridges, can also be prepared by genetic engineering when a gene which codes for a polypeptide of the formula I or part sequences thereof is incorporated in an expression plasmid. This polypeptide is also called "hirudin" in the following text.

The requisite gene can be chemically synthesized by known methods, isolated from the genome and processed, or the mRNA can be isolated from induced cells by known methods and the cDNA can be obtained from this.

The route via cDNA is preferred, and especially chemical synthesis, in particular by the phosphite method. It is furthermore preferred to synthesize a gene which codes for the polypeptide of the formula I, in which m is 1 and n is zero, X represents Met or Arg, B represents Thr or a direct bond, C represents Thr, and G represents Glu. Another preferred embodiment of the invention relates to polypeptides of the formula I, in which (X)m-A-B-C- does not represent Val-Val- when n is zero, D and E denote Thr, F denotes Gly, and G and H denote Glu.

It is particularly preferred to synthesize polypeptides of the formula I, in which m is 1, X denotes Met or Arg, A and B denote direct bonds, C, D and E denote Thr, F denotes Gly, G and H denote Glu, and n denotes zero. The DNA sequence I which is preferred for this purpose is shown in the attachment and is used in the following text to illustrate the invention.

It is known that the genetic code is "degenerate", i.e. only two amino acids are coded for by a single nucleotide sequence, whereas 2 to 6 triplets are assigned to the remaining 18 genetically codable amino acids. Moreover, the host cells of different species do not always make the same use of the possibilities of variation resulting from this. Thus, there is an immense variety of codon possibilities for the synthesis of the genes.

It has now been found that the DNA sequence I (attachment), which codes for the entire amino acid sequence, and the DNA part sequences II A and II B, which are used for the synthesis of sequence I, are particularly advantageous for the genetic engineering synthesis of the particularly preferred form of hirudin. A "protruding" DNA sequence corresponding to the restriction endonuclease Xbal is located at the 5' end of the coding strand of DNA sequence I, whereas the single-stranded protruding sequence corresponding to the restriction enzyme Sal I is located at the 3' end of the 4 coding strand. These two different recognition sequences ensure the insertion of the DNA in plasmids in the desired orientation. (However, it is also possible to select identical recognition sequences and to undertake appropriate selection after characterization of the plasmid DNA by appropriate restriction or DNA sequence analysis or by expression).

Between these recognition sequences and the codons for the amino acid sequence is located at the 5' end of the coding strand the codon for the amino acid methionine (which is numbered 0 in DNA sequence I). This can be replaced by a presequence (also called a signal or leader sequence) of a bacterial or other host-intrinsic protein (review article: Perlman and Halvorson; J. Mol. Biol. 167 (1983), 391), which brings about the secretion of the desired polypeptide from the cytoplasm, and in this excretion process is split off by a signal peptidase naturally occurring in the host cell. Then, according to the invention, at the end of the coding strand the triplet coding for glutamine is followed by a stop codon or, preferably, as shown in DNA sequence I, two stop triplets. In addition, a nucleotide sequence corresponding to the restriction enzyme SstI is incorporated in DNA sequence I between these stop codons and the protruding Sail end.

A unique internal cleavage site for the restriction enzyme Bam HI (in codon 30/31) permits the subcloning of two gene fragments HIR-I and HIR-II (see DNA sequence II), which can be incorporated into cloning vectors which have been thoroughly investigated, such as, for example, pBR 322, pUC 8 or pUC 12. In addition, a number of other unique recognition sequences for restriction enzymes are incorporated within the gene, and these provide, on the one hand, access to part sequences of hirudin and permit, on the other hand, variations to be carried out (Table 1): 5 Table 1: Restriction Cleavage after nucleotide No. enzyme (coding strand) Rsa I«) 8 5 Acc I 12 Hinf I 27 Xho IIa) 57 Fnu 4 HI 71 Hae III 73 10 Bst NI 75 Hph I 117 Rsa Ib) 137 Kpn I 139 Taq I 172 15 Xho Hb) 178 Dde I 184 Mbo II 186 Sst I 210 a) unique with respect to part sequence HIR-I 20 b) " M " " " " HIR-II DNA sequence I can be synthesized from 14 oligonucleotides with lengths of from 25 to 35 nucleotides (see DNA sequence II), by first chemically synthesizing them and then linking them enzymatically via "sticky 25 ends" of 4 to 6 nucleotides.

In addition, in DNA sequence I account has been taken of the fact that for those amino acids to which several codons are assigned the latter are not equivalent but, on the contrary, they show different preferences in the 30 particular host cell, such as E. coli. Furthermore, palindromic sequences have been reduced to a minimum.

Thus, the gene structure of DNA sequence I is readily accessible from relatively small units, it makes possible the subcloning of two gene fragments into well-known 6 vectors, and it permits their combination to give the total gene and, where appropriate, modifications thereof.

Depending on the incorporation of the synthetic gene into the cloning vector, the desired peptide having the amino acid sequence of hirudin is either expressed directly (DNA sequence IC) or is expressed in the form of a fusion protein with a bacterial protein such as 0-galactosidase or partial sequences thereof. These fusion proteins can then be chemically or enzymatically cleaved in a known manner, if, for example, the amino acid methionine (DNA sequence IA) is located in position 0 of sequence I, chemical cleavage with cyanogen bromide can be carried out, and if the amino acid arginine (DNA sequence IB) is located in this position, then enzymatic cleavage with trypsin can be carried out.

Preferred variations of the amino acid sequences are shown in Table II, m and n being zero: Table 2: A B C D E F G H a) lie Thr Thr Thr Thr Gly Glu Glu b) lie - Thr Thr Thr Gly Glu Glu c) lie - Thr Thr lie Gly Glu Glu d) lie - Thr Thr Thr Gly Glu Pro e) lie - Thr Thr lie Gly Glu Pro f) - - Thr - Thr Gly - Glu g) - - Thr Thr Thr Gly — Glu Depending on the composition of the synthetic gene, these modifications can readily be brought about at the DNA level by replacement of the appropriate gene fragments by de-novo-synthesized DNA sequences, utilizing appropriate restriction enzyme cleavage sites.

An example of a variation of the amino acid sequence which may be mentioned is the (known) polypeptide of the formula I, in which m is 1, X and C represent Val, A and B represent direct bonds, D and E denote Thr, F denotes ? Gly, G and H denote Glu, and n denotes zero. This modification can readily be brought about at the DNA level via the cleavage site corresponding to the restriction enzyme Acc I.

The incorporation of the synthetic genes or the gene fragments into cloning vectors, for example into the commercially available plasmids pUC 8, pUC 12 and pBR 322, or other generally accessible plasmids, such as ptac 11 and pKK 177.3, is carried out in a manner known per se. It is also possible beforehand to provide the chemically synthesized genes with suitable chemically synthesized control regions which make expression of the proteins possible. In this context, reference may be made to the textbook by Maniatis (Molecular Cloning, Maniatis et al., Cold Spring Harbor, 1982). The transformation into suitable host organisms, advantageously E. coli, of the hybrid plasmids thus obtained is likewise known per se and is described in detail in the abovementioned textbook. The isolation of the expressed protein and its purification can be carried out by processes known per se.

The gene fragments HIR-I and HIR-II obtained according to the invention, the hybrid plasmids obtained therewith, and the transformed host organisms are new and the invention relates to them. The same applies to the new DNA sequences which are modifications of DNA sequence I. Further embodiments of the invention are set out in the patent claims.

Some other embodiments of the invention are illustrated in detail in the examples which follow, from which the multiplicity of the possible modifications and combinations are evident to those skilled in the art. In these examples, percentage data relate to weight unless otherwise specified.

Examples 1. Chemical synthesis of a single-stranded oligonucleotide The synthesis of the structural units of the gene is illustrated using the example of structural unit la of the gene, which comprises nucleotides 1-32 of the coding strand. The nucleoside at the 3' end, in the present case thymidine (nucleotide No. 32), is covalently bonded via the 3'-hydroxyl group by known methods (M. J. Gait et al., Nucleic Acids Res. 8 (1980) 1081-1096) to silica gel ((R)FRACTOSIL, supplied by Merck). For this purpose, initially the silica gel is reacted with 3-(triethoxysilyl)propylamine with elimination of ethanol, an Si-O-Si bond being produced. The thymidine is reacted as the 3 '-0-succinoyl-5'-dimethoxytrityl ether with the modified carrier in the presence of paranitrophenol and N,N'-dicyclohexylcarbodiimide, the free carboxyl group of the succinoyl group acylating the amino radical of the propylamino group.

In the following synthesis steps, the base component is used as the monomethyl ester dialkylamide or chloride of the 5'-0-dimethoxytritylnucleoside-3'-phosphorus acid, the adenine being in the form of the N6-benzoyl compound, the cytosine in the form of the NA-benzoyl compound, the guanine in the form of the N2-isobytyryl compound, and the thymine, which contains no amino group, being without a protective group. 50 mg of the polymeric carrier which contains 2 /imol of bound thymidine are treated successively with the following agents: a) nitromethane, b) saturated zinc bromide solution in nitromethane containing 1% water, c) methanol, d) tetrahydrofuran, 9 e) acetonitrile, f) 40 fimol of the appropriate nucleoside phosphite and 200 /jmol of tetrazole in 0.5 ml of anhydrous acetonitrile (5 minutes), g) 20% acetic anhydride in tetrahydrofuran containing 40% lutidine and 10% dimethylaminopyridine (2 minutes), h) tetrahydrofuran, i) tetrahydrofuran containing 20% water and 40% lutidine, j) 3% iodine in collidine/water/tetrahydrofuran in the ratio by volume 5:4:1, k) tetrahydrofuran, and 1) methanol.

The term "phosphite" in this context is to be understood to be the monomethyl ester of the deoxyribose-3'-monophosphorous acid, the third valency being saturated by chlorine or a tertiary amino group, for example a morpholino radical. The yields of the individual synthesis steps can be determined in each case after the detritylation reaction b) by spectrophotometry by measurement of the absorption of the dimethoxytrityl cation at a wavelength of 496 nm.

When the synthesis of the oligonucleotide is complete, the methyl phosphate protective groups of the oligomer are eliminated using p-thiocresol and triethylamine.

The oligonucleotide is then separated from the solid carrier by treatment with ammonia for 3 hours. Treatment of the oligomers with concentrated ammonia for 2 to 3 days quantitatively eliminates the amino protective groups on the bases. The crude product thus obtained is purified by high-pressure liquid chromatography (HPLC) or by polyacrylamide gel electrophoresis.

The other structural units Ib-IIh of the gene are also synthesized entirely correspondingly, their nucleotide I 0 sequences being evident from DNA sequence II. 2. Enzymatic linkage of the single-stranded oligonucleotides to give the gene fragments HIR-I and HIR-II For the phosphorylation of the oligonucleotides at the 5' terminal end, 1 nmol of each of oligonucleotides Ib-Ie was treated with 5 nmol of adenosine triphosphate, with four units of T4 polynucleotide kinase in 20 pi of 50 mM tris.HCl buffer (pH 7.6), 10 mM magnesium chloride and 10 mM dithiothreitol (DTT), at 37°C for 30 minutes. The enzyme is inactivated by heating at 95°C for 5 minutes. The oligonucleotides la, If, Ila and Ilh, which form the "protruding" sequence in DNA sequences IIA and I IB, are not phosphorylated. This prevents, in the subsequent ligation, the formation of larger subfragments than correspond to DNA sequence IIA or I IB.

The oligonucleotides Ia-If are ligated to form the subfragment HIR-I as follows: 1 mmol of each of the oligonucleotides la and If and of the 5'-phosphates of lb, Ic, Id and Ie are dissolved together in 45 n 1 of buffer containing 50 mM tris.HCl (pH 7.6), 20 mM magnesium chloride, 25 mM potassium chloride and 10 mM DTT. For the annealing of the oligonucleotides corresponding to DNA sequence IIA, the solution of the oligonucleotides is heated at 95°C for 2 minutes and then slowly (2-3 hours) cooled to 20°C. Then, for the enzymatic linkage, 2 ^1 of 0.1 M DTT, 8 n 1 of 2.5 mM adenosine triphosphate (pH 7) and 5 pi of T4 DNA ligase (2,000 units) are added, and the mixture is incubated at 22°C for 16 hours.

The oligonucleotides lib to Ilg are phosphorylated and then ligated together with the oligonucleotides Ila and Ilh to give the subfragment HIR-II analogously.

The gene fragments HIR-I and HIR-II are purified by gel electrophoresis on a 10% polyacryl amide gel (without addition of urea, 20 x 40 cm, 1 mm thick), the marker substance used being $X 174 DNA (supplied by BRL) cut with Hinf I, or pBR 322 cut with Hae III. 3. Preparation of hybrid plasmids which contain the gene fragments HIR-I and HIR-II a) Incorporation of the gene fragment HIR-I in pUC 12 The commercially available plasmid pUC 12 is opened in a known manner using the restriction endonucleases Xba I and Bam HI in accordance with the manufacturer's instructions. The digestion mixture is fractionated on a 5% polyacrylamide gel by electrophoresis in a known manner, and the fragments are visualized by staining with ethidium bromide or by radioactive labeling ("nick translation", cf. Maniatis, loc. cit.). The plasmid band is then cut out of the acrylamide gel and separated from the polyacrylamide by electophoresis. The fractionation of the digestion mixture can also be carried out on 2% low-melting agarose gels (as described in Example 5a)). 1 ng of plasmid is then ligated at 16 °C overnight with 10 ng of gene fragment HIR-I which has previously been phosphorylated as described in Example II. The hybrid plasmid as shown in Figure I is obtained. b) Incorporation of the gene fragment HIR-II in pUC 8 In analogy to a), the commercially available plasmid pUC 8 is cut open with Bam HI and Sal I and ligated with the gene fragment HIR-II which has previously been phosphorylated as described in Example 2. The hybrid plasmid as shown in Figure 2 is obtained. 1 2 Synthesis of the complete gene and incorporation in a plasmid Transformation and amplification The hybrid plasmids thus obtained are transformed into E. coli. For this purpose,"the strain E. coli R 12 is made competent by treatment with a 70 mM calcium chloride solution, and the suspension of the hybrid plasmid in 10 mM tris.HCl buffer (pH 7.5), which is 70 mM in calcium chloride, is added. The transformed strains are selected as usual utilizing the resistance and sensitivity to antibiotics conferred by the plasmid, and the hybrid vectors are amplified. After the cells have been killed , the hybrid plasmids are isolated, cut open with the originally used restriction enzymes, and the gene fragments HIR-I and HIR-II are isolated by gel electrophoresis.

Linkage of the gene fragments The subfragments HIR-I and HIR-II obtained by amplification are enzymatically linked as described in Example 2 (annealing at 60#C), and the synthetic gene with the DNA sequence I which has been thus obtained is introduced into the cloning vector pUC 12. A hybrid plasmid as shown in Figure 3 is obtained.

Preparation of a gene for [Val1, Val2] -hirudin The Accl-Sall fragment (nucleotides 13-212) is obtained as described in Example 5a from the plasmid as shown in Figure 3, and this fragment can then be ligated, using the following adaptor 5' CT AGA ATG GTT GTA T 3' 3' T TAC CAA CAT ATA 5' Xbal AccI into the plasmid pUC 12 which has previously been 1 3 opened with Xbal and Sail.

Preparation of a gene for variant a) in Table 2 The following DNA sequence is synthesized for this purpose: Glu Phe Met lie Thr Thr 5' GG GAA TTC ATG ATC ACA ACG T 3' CC CTT AAG TAC TAG TGT TGC ATA Sraal AccI This sequence can be incorporated as a Smal-AccI adaptor into the DNA from the plasmid as shown in Figure 3, which has been opened with the restriction enzymes Smal and AccI.

Preparation of a gene for variant b) in Table 2 The following DNA sequence is first synthesized: Glu Phe Met lie Thr 5' GG GAA TTC ATG ATC ACG T 3' CC CTT AAG TAC TAG TGC ATA SmaI AccI This DNA sequence which has been shortened by the ACA1triplet is incorporated as described under d).

Preparation of a gene for variant c) in Table 2 The starting DNA sequence is that previously described under e) (variant b) in Table 2). After cleavage with AccI and Sail, two fragments are obtained and isolated. The Accl-Sall-(hirudin) fragment is cut with the enzyme Hinfl, and the larger of the fragments obtained is isolated. This fragment, the residual plasmid DNA opened with Accl/Sall, and the synthetic DNA sequence Thr Asp Cys lie 5' AT ACT GAC TGC ATC G 3» TGA CTG ACG TAG CTT A AccI Hlnfl are ligated in the same vessel. The resulting hybrid \ 4 plasmid codes for variant c) in Table 2.

Preparation of a gene for variant d) in Table 2 The starting DNA sequence is again that previously described under e) and thus codes for variant b) in Table 2, this sequence being cut with AccI and Sail. The AccI-SalI-(hirudin) fragment is, however, reacted with TaqI and Ddel, and the fragment which has been cut out is replaced by the synthetic DNA sequence Glu Pro lie 5' C GAA CCG ATC CC 3' TT GGC TAG GGA CT TaqI Dde I The AccI-Sall-(hirudin) fragment which has thus been modified is now ligated with the residual plasmid DNA. The resulting plasmid codes for variant d) in Table 2.

Preparation of a gene for variant g) in Table 2 The BamHI-SalI-(hirudin) fragment is isolated from the hybrid plasmid as shown in Figure 2, and is then cut with KpnI. The larger of the two resulting fragments is isolated, and is ligated with the synthetic DNA sequence Ser Asp Gly Lys Asn Gin 5' GA TCC GAC GAA AAG AAC CAG G CTG CTT TTC TTG GTC BamHI Thr Gly Glu Gly 5* ACT GGC GAA GGT AC 3' TGA CCG CTT C Kpn I The ligation product is now linked with the Xbal-BamHI-(hirudin) fragment isolated from the plasmid DNA shown in Figure 1, and is introduced into pUC 12 as described under 4b). The hybrid plasmid which Cys Val TGC GTT ACG CAA 1 5 is obtained codes for variant g) in Table 2. Preparation of a gene for variant f) in Table 2 The XbaI-SalI-(hirudin) fragment is isolated from the DNA which codes for variant g) in Table2, and is reacted with Hinfl. The larger of the two resulting fragments is then ligated with the synthetic DNA sequence Arg Met Thr Tyr Asp Cys Thr 5' CT AGA ATG ACG TAT GAC TGC ACT G 3' T TAC TGC ATA CTG ACG TGA CTT A Xbal Hinf I and the ligation product is inserted into the plasmid pUC 12 which has been opened with Xbal and 10 Sail. The hybrid plasmid codes for variant f) in Table 2. j) Preparation of other variants The following may be mentioned as examples of other possible variants: The DNA sequences which code for variants c) and d) in Table 2 contain the BamHI recognition site corresponding to position 97 bp of DNA sequence I. Now, utilizing this cleavage site, it is possible to link together as desired the AccI-BamHI and BamHI-Sall part sequences of the two variants which have been described above.

Transformation of all these variants into E. coli and their expression is possible in analogy to the descriptj on for DNA sequence I. 25 5. Construction of hybrid plasmids for the expression of DNA sequences IA, IB and IC a) Incorporation of DNA sequence IC in pKK 177.3 i) 5 15 20 1 6 (direct expression) The expression plasmid pKK 177.3 (plasmid ptac 11, Amman et al., Gene 25 (1983) 167, into the Eco RI recognition site of which a sequence which contains a Sal I cleavage site has been incorporated synthetically) is opened with the restriction enzymes Eco RI and Sal I. The DNA sequence IC is obtained from the plasmid as shown in Figure 3 as follows. The plasmid is cut first with the restriction enzyme Sal I and then with the enzyme Acc I, and then the small Acc I-Sal I fragment is separated from the plasmid band by gel electrophoresis on a 2% low-melting agarose gel, the DNA being recovered by dissolution of the gel at elevated temperature (as stated by the manufacturer). This DNA fragment can be converted into DNA sequence IC using the following adaptor 5' AATTC ATG ACG T 3' 3' G TAC TGC ATA 5 * Eco RI Acc I A hybrid plasmid in which there is an expression or regulation region upstream of the insert is produced by ligation of the plasmid pKK 177.3, which has been cut open, with the gene IC. After addition of a suitable inducer, such as isopropyl p-thiogalactopyranoside (IPTG), a mRNA which leads to expression of the methionyl-polypeptide corresponding to DNA sequence IC is formed.

Incorporation of the hirudin genes IA and IB into the expression plasmid pCK-5196 (fusion construction) The expression plasmid pCK-5196 (Fig. 4) is a derivative of the plasmid pAT 153 (Twigg and Sherrat) which itself is a high copy-number derivative of the plasmid pBR 322. It contains the known Lac UV5 promoter with the known sequence of p-galactosidase up to nucleotide No. 1554 1 7 (corresponding to amino acid No. 518:Trp) inserted between the tetR gene (Hind III from pBR 322) and the terminator of the ampR gene (Aha III in Pos. 3231 in pBR 322). Another feature of this vector is the polylinker sequence which originates from the known plasmid pUC 13 and is inserted between the 0-galactosidase sequence and the terminator sequence. Transcription from the lac UV5 promoter takes place counter to the tetR gene.

This plasmid contains at amino acid No. 518 of ^-galactosidase the polylinker portion Xba I-Bam HI-Sma I-Sst I from the plasmid pUC 13, which serves as the cloning site for eucaryotic genes. The plasmid pCK-5196 is opened using the restriction enzymes Xba I and Sst I and is ligated with DNA sequence IA which can be isolated from the plasmid shown in Fig. 3 by Xba I/SSt I digestion. A hybrid plasmid as shown in Fig. 5 is obtained, and this contains downstream of the lac UV5 control region the codons for the first 518 amino acids of /9-galactosidase followed by the codons for Ser-Arg-Met-(hirudin 1-64) .

A hybrid plasmid as shown in Fig. 6 can be obtained in the same manner, this containing in the plasmid pCK-5196 an insert of DNA sequence IB in which, however, the codon for amino acid No. 1 (threonine) is now preceded by the codon for the amino acid arginine. The DNA sequence IB is obtained from the plasmid shown in Fig. 3 by cutting the latter with the restriction enzymes Sst I and Acc I and ligating with the following adaptor: 5* CT AGA CGT ACG T 3' 3' T GCA TGC ATA 5' Xba I Acc I 1 8 Transformation of the hybrid plasmids.

Competent E. coli cells are transformed with 0.1 to 1 jig of the hybrid plasmids which contain sequence I, or IA, IB or IC, and in the case of tac plasmids are plated out onto agar plates containing ampicillin, and in the case of the pCK-5196 hybrid plasmids are plated out onto agar plates containing tetracycline. Clones which contain the correctly integrated hirudin sequences in the particular plasmids can then be identified by DNA rapid work-up (Maniatis, loc. cit.).

Expression of the polypeptides having hirudin activity Following transformation of the specified tac hybrid plasmids into E. coli., the polypeptide expressed carries, in addition to the hirudin amino acid sequence, an additional methionyl group at the amino terminal end, but this group can be eliminated by cleavage with cyanogen bromide. On the other hand, following transformation of E. coli with the hybrid plasmid shown in Fig. 5 or Fig. 6, the fusion proteins obtained comprise 518 amino acids of p-galactosidase and hirudin which are linked together via the sequence Ser-Arg-Met or Ser-Arg-Arg. These fusion proteins can be cleaved to give hirudin and j9-galactosidase fragments using cyanogen bromide or trypsin.

Working up and purification The bacterial strains which have been cultivated to the desired optical density are incubated for a sufficient time, for example 2 hours, with a suitable inducer, for example IPTG. The cells are then killed with 0.1% cresol and 0.1 mM benzylsulfonyl fluoride.

J 9 After centrifugation or filtration, the mass of cells is taken up in a buffer solution (50 mM tris, 50 mM EDTA, pH 7.5) and is disrupted mechanically, for example using a French press or (R)DYN0 mill (supplied by Willy Bachofer, Basle), whereupon the insoluble constituents are removed by centrifugation. The protein containing the hirudin is purified from the supernatant by customary processes. Ion exchange, adsorption, or gel filtration columns or affinity chromatography on thrombin or antibody columns are suitable. The enrichment and purity of the product are checked by analysis using sodium dodecyl sulfate/acrylamide gels or HPLC. Fusion proteins containing a p-galactosidase fraction can be detected even in the crude extract of the lysed bacteria on the basis of the difference in their migration behavior from that of 0-galactosidase (1-518). The hirudin produced by genetic engineering is characterized by a comparison with the authentic substance isolated from leeches or by means of tests based on the anticoagulant properties of hirudin. 20 DNA seauence I Triplet No. 0 1 2 3 4 5 Amino acid Met Thr iyr Thr Asp Cys Nucleotide No. 1 10 20 Cod. st rand 5' CT AGA ATG ACG TAT ACT GAC TGC Non- ■cod. strand 3' T TAC TGC ATA TGA CTG ACG 6 7 8 9 10 11 12 13 14 15 Thr Glu Ser Gly Gin Asn Leu Cys Leu Cys 30 40 50 ACT GAA TCT GGT CAG AAC CTG TGC CTG TGC TGA CTT AGA CCA GTC TTC GAC ACG GAC ACG 16 17 18 19 20 21 22 23 24 25 Glu Gly Ser Asn Val Cys Gly Gin Gly Asn 60 70 80 GAA GGA TCT AAC GTT TGC GGC CAG GGT AAC CTT CCT AGA TTG CAA ACG CCG GTC CCA TTG 26 27 28 29 30 31 32 33 34 35 Lys Cys lie Leu Gly Ser Asp Gly Glu Lys 90 100 110 AAA TGC ATC CTT GGA TCC GAC GGT GAA AAG TTT ACG TAG GAA CCT AGG CTC CCA CTT TTC 36 37 38 39 40 41 42 43 44 45 Asn Gin Cys Val Thr Gly Glu Gly Thr Pro 120 130 140 AAC CAG TO C GTT ACT GGC GAA GGT ACC CCG TTG GTC ACG CAA TGA CCG CTT CCA TGG GGC 46 47 48 49 50 51 52 53 54 55 Lys Pro Gin Ser His Asn Asp Gly Asp Phe 150 160 170 AAA CCG CAG TCT CAT AAC GAC GGC GAC TTC TTT GGC GTC AGA GTA TTG CTG CCG CTG AAG 56 57 58 59 60 61 62 63 64 Glu Glu lie Pro Glu Glu Tyr Leu Gin Stp 180 190 200 GAA GAG ATC CCT GAG GAA TAC CTT CAG TAA CTT CTC TAG GGA CTC CTT ATC GAA GTC ATT Stp 210 TAG AX TCG 3' ATC TCG AGC AGC T 5' 2-t DNA sequence I A: 0 Met (Amino acids 1-64) 1 5 205 210 5' CT AGA ATG (Nucleotides 9-200) TAA TAG AGC T 3' 3' T TAC (Complement, nucl.) ATT TAC 51 Xbal SstI DNA sequence IB: 0 Arg (Amino acids 1-64) 1 5 205 210 5' CT AGA CGT (Nucleotides 9-200) TAA TAG AGC T 3' 3' T TAC (Complement, nuc I.) ATT ATC 5' Xbal SstI DNA sequence IC: O Met (Amino acids 1-64) 205 210 5' AA TTC ATG (Nucleotides 9-200) TAA TAG AGC TCG 3' G TAC (Complement, nucl.) ATT ATC TCG AGC AGC T 5' EcoRI Sail DNA sequence II A (Hlft-I) I a Nucleotide No. 20 20 Cod. strand 5, CT AGA aTG ACG TAT ACT GAC Non-cod. strand 31 JAC TGC ATA TGA CTG Xbal I b I c 30 40 50 GAA TCT GGT CAG AAC CTG TGC CTG TGC CTT AGA CCA GTC TTG GAC ACG GAC ACG ACT TGA I b I c I d I e 60 70 80 GAA GGA TCT AAC GTT TGC GGC CAG GGT AAC CTT CCT AGA TTG CAA ACG CCG GTC CCA TTG I d I e 90 AAA TGC ATC CTT G Bam HI 3' TTT ACG TAG GAA CCT AG 5' I f If DNA sequence II 8 (HIR-II) Nucleotide No. Cod. st rand Non-cod. strand II a 120 AAC CAG TGC TTG GTC ACG II b II c 150 AAA CCG CAG TTT GGC GTC II d II e 180 GAA GAG ATC CTT CTC TAG II f II g 210 TAG AGC TCG ATC TCG AGC II h II a 100 110 5' GA TCC GAC GGT GAA AAG 3' BamHI G CTG CCA CTT TTC II b II c 130 140 GTT ACT GGC GAA GGT ACC CCG CAA TGA CCG CTT CCA TGG GGC II d I e 160 170 TCT CAT AAC GAC GGC GAC TTC AGA GTA TTG CTG CCG CTG AAG II f II g 190 200 CCT GAG GAA TAC CTT CAG TAA GGA CTC CTT ATG GAA GTC ATT Ilh Sal I 3' AGC T 5' 24

Claims (12)

CLAIMS:

1. A process for the preparation of a polypeptide having hirudin activity, of the formula I (X)m.1-X,-OTyr-D-Asp-Cys-E-Gtu-S©r-Gly-GlrvAsrvLeu-Cys-Let>Cys-GlLK3(y-S€r-5 Asn-Val-Cys-Gly-Gln-Gly^AsrvLys-Cys-lte-LeiiGty-Ser-Asp-F-^-Lys-Asrv-Gln-Cys- 40 45 50 55 Val-Thr-Gty-Glu-Gly-Thr-Pro-Lys-Pro-GlrvSer-His-AsrvAsp-GJy-Asp-Phe-Glu-H-lle- Pro-Glu-Glu-Tyr-Leu-Gln-(Z)n-OH in which m = o in I o 10 n = 0 - 100, and X and X1 represent identical or different residues of genetically codable amino acids, C denotes Thr, Val, lie, Leu or Phe, D denotes Thr, 15 E denotes Thr, F denotes Gly, G denotes Glu, H denotes Glu, and Z represents identical or different residues of 20 genetically codable amino acids, which comprises incorporation into an expression plasmid of a gene which codes for a polypeptide of the formula I, it being possible for other amino acids to be omitted or replaced by (an)other genetically codable amino acid(s), excepting the preparation of the following polypeptides in which a) X1 and C are both Val or b) X1 denotes lie and C denotes Thr or c) N-terminal amino acids are eliminated up to position 30 10-

2. The process as claimed in claim 1, wherein the non-coding strand of the incorporated gene hybridizes with the coding strand of the gene coding for the polypeptide of the formula I.

3. The process as claimed in claim 1 or 2, wherein the gene has been synthesized chemically/ preferably by the phosphite process.

4. The process as claimed in one or more of the preceding claims, wherein the gene contains two stop codons downstream of the codon for the carboxy-terminal amino acid.

5. The process as claimed in one or more of the preceding claims, wherein the gene corresponds to the DNA sequence I in Table 1.

6. DNA sequences I, IA, IB, IC, IIA and I IB corresponding to Tables 1 to 4.

7. A fusion peptide which contains a polypeptide as claimed in claim 1, and wherein its bacterial fraction preferably contains all or part of the amino acid sequence of 0-galactosidase.

8. A hybrid plasmid which contains a DNA sequence which codes for the polypeptide of the formula I.

9. A host organism, preferably E. coli, which contains a hybrid plasmid as claimed in claim 8.

10. A process as claimed in claim 1, substantially as hereinbefore described and exemplified.

11. A polypepide of the formula I given and defined in claim 1, having hirudin activity, whenever prepared by a process claimed in a preceding claim.

12. A fusion peptide as claimed in claim 7, substantially as hereinbefore described and exemplified. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.