FI97239B - Process for the preparation of fusion protein - Google Patents

Description

97239

Method for preparing a fusion protein

Divided separated from application 925312.

The invention relates to a process for the preparation of a fusion protein having both interleukin-2 and hirudin activity. Also described is the "open reading frame" of DNA encoding interleukin-2 and the use of this DNA as an expression aid to produce peptides or proteins.

10 In the production of eukaryotic proteins, the gene is technically often obtained in bacteria in low yields, especially in the case of small proteins with a molecular weight of up to about 15,000 daltons and a structure containing disulfide bridges. It is hypothesized that 15 of the host's own proteases rapidly degrade the proteins formed. Therefore, it is expedient to generate gene constructs encoding fusion proteins in which the undesired part of the fusion protein is the host's own protein, which is removed after isolation of the primary product by methods known per se.

It has now surprisingly been found that the N-terminal part of interleukin-2, corresponding approximately to the first 100 amino acids, is particularly well suited for the preparation of fusion proteins. Thus, the primary product is: a fusion protein consisting wholly or very largely of eukaryotic protein sequences. Surprisingly, the host organism in question apparently does not recognize this protein as a foreign protein and does not immediately degrade again. A further advantage is that the fusion proteins of the invention are sparingly soluble or insoluble, and can thus be separated from the soluble proteins simply by convenient centrifugation. Since the function of the interleukin-2 moiety as a "ballast portion" of the fusion protein is not dependent on the fact that this moiety 35 is a biologically active molecule, there is also no dependence on the exact structure of the interleukin-2 moiety. For this purpose, it is sufficient that approximately the first 100 N-terminal amino acids are present. Thus, for example, it is possible to make changes at the N-terminus that allow cleavage of the fusion protein if the desired protein is N-terminally associated with it. Likewise, changes can be made to the C-terminus to allow or facilitate cleavage of the desired protein when it is - as usual - associated with the 10 C-terminus of the fusion protein.

The natural DNA sequence encoding human interleukin-2, hereinafter "IL-2", is described in EP-A-0 091 539. The literature therein deals with murine and rat IL-2. This mammalian DNA can be used in accordance with the invention to synthesize proteins. However, it is more expedient to start from synthetic DNA, particularly preferably from the corresponding DNA of human IL-2, as proposed in (unpublished) DE-A-3 419 995 (corresponding to EP-A-0 163 249). 20 This synthetic DNA sequence is given at the end of the explanatory note in Annex I (DNA sequence I). This synthetic DNA sequence has the advantage not only of being suitable for the most frequently used host of codon selection, E. coli, but also of containing a number of: restriction endonuclease cleavage sites that can be utilized in accordance with the invention. Table 1 below shows a selection of suitable cleavage sites from the beginning of the cycle or from the 100th triplet region. However, this does not preclude making DNA changes in the intervening region, in which case the other cleavage sites provided in the above-mentioned patent application can be exploited.

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table 1

Restriction Identification Enzyme Sequence of the Identification Sequence First Nucleotide Position (Coding Thread) 5 5 '3'

Aha II, Ban I,

Hae II, Nar I, GGCGCC 8

Ban II, Sac I,

Sst I GAGCTC 291 10 Hha I GCGC 9

Hinf I GACTC 35

Pvu I CGATCG 346

Tag I_TCGA_387_ 15

If the nucleases BanII, SacI or SStI are used, an IL-2 subsequence encoding about 95 amino acids is obtained. This length is generally sufficient to obtain an insoluble fusion protein. If the low solubility, for example in the case of the desired hydrophilic eukaryotic protein, is not yet sufficient and it is not desired to use cleavage sites closer to the C-terminus to provide the lowest possible "ballast", the DNA sequence may be extended from the N- and / or C-terminus. with a suitable adapter, i.e. a coupler, and "tailor" the "ballast" part in that way. Of course, a DNA sequence can also be used - more or less to the end, thus providing a potentially modified biologically active IL-2 as a "by-product" or a bifunctional protein that has IL-2 activity in addition to the encoded protein.

The present invention relates to a method for producing a fusion protein, which method is characterized by expressing in a bacterial host cell a gene encoding a C- or N-terminal part substantially corresponding to the amino acid sequence of interleukin-2 and further encoding hirudin.

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Preferably, a gene encoding a fusion protein of general formula is used

Met - X - Y - Z or Met - Z - Y - X

5 (Ia) (Ib) wherein X substantially corresponds to the amino acid sequence of human IL-2, Y is a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows cleavage of the desired 10 protein, or otherwise a bridging sequence of one or more genetically encodable amino acids , which allows cleavage, and Z is the amino acid sequence of hirudin.

As shown in formulas Ia and Ib - and as already mentioned above - it is possible to cause the protein to be expressed before or after the IL-2 moiety. For the sake of simplicity, the first possibility, which corresponds to the general method for the production of fusion proteins, is mainly illustrated below. Thus, when this "classic" 20 option is described below, the second option is thus not excluded.

The cleavage of the fusion protein can be performed chemically or enzymatically in a manner known per se. The choice of a suitable method depends above all on the amino acid sequence of the desired protein. For example, if this does not contain methionine, Y may be Met, which undergoes chemical cleavage with chlorine or bromo-cyano. If the carboxyl terminus of the linker Y is cysteine or Y is Cys, enzymatic cysteine-specific cleavage or chemical cleavage may be performed, for example, after specific S-cyanolation. If the carboxyl terminus of the linking moiety Y is tryptophan or Y is Trp, chemical cleavage with N-bromosuccinimide can be performed.

Proteins whose amino acid sequence does not contain the pair 35 Asp-Pro 5 97239 and which are sufficiently acid stable can be proteolytically cleaved in a manner known per se. This results in proteins containing an N-terminal proline group or a C-terminal aspartic acid group. Thus, modified proteins can also be synthesized in this way.

The Asp-Pro bond can be made even more sensitive to acids when this intermediate is (Asp) n-Pro or Glu- (Asp) n-Pro, where n is 1 to 3.

Examples of enzymatic cleavages are also known, in which modified enzymes with improved specificity can also be used [cf. C.S. Craik et al., Science 228 (1985) 291-297]. If the desired eukaryotic peptide is proinsulin; it is appropriate to select as the sequence Y a peptide sequence in which the trypsin-cleavable amino acid (Arg, Lys) is linked to the N-terminal amino acid (Phe) of proinsulin, for example the sequence Ala-Ser-Met-Thr-Arg, because then arginine-specific cleavage by trypsin protease.

20 Unless the desired protein contains an amino acid sequence

Ile-Glu-Gly-Arg, can be a fusion protein silicon with factor Xa (EP-: 25 applications 0 025 190 and 0 161 973).

The fusion protein is formed in a manner known per se through expression in a suitable expression system. All known host vector systems are suitable for this purpose, i.e. for example mammalian cells and microorganisms, for example yeasts and preferably bacteria, in particular E. coli.

The DNA sequence encoding the desired protein is inserted in a known manner into a vector that provides good expression in the selected expression system.

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In the case of bacterial hosts, the promoter and operator are suitably selected from the group consisting of Lac, tae, trp, λ-phage PL or P ', hsp, omp or a synthetic promoter, which are proposed, for example, in DE-A-3 430 683 (EP-A-0 173 149 ). The preferred promoter-operator sequence is a guarantee which, moreover, is commercially available (e.g. the expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, p. 64).

In the context of expression of a fusion protein of the invention, it may be appropriate to modify the individual triplets corresponding to the first amino acids following the ATG start codon to prevent possible base pairing at the mRNA level. Such alterations, as well as alterations, deletions, or additions of individual amino acids in the IL-2 protein moiety, are within the skill of the art and are within the scope of the invention.

The invention is illustrated in more detail in the following examples and drawings. Thus, Figure 1 and its extension, Figure 1a, show the synthesis of plasmid pK360, which plasmid encodes a fusion protein containing a hirudin sequence, Figure 2 and its extension, Figure 2a, show the synthesis of plasmid pK410, which plasmid likewise encodes a fusion protein containing a hirudin amino acid sequence, Figure 3 and its extensions, Figures 3a to 3c, show the construction of plasmids pPH15, 16, 20 and 30, which plasmids encode plasmid fusion proteins containing the amino acid sequence of monkey proHI insulin, which encodes a fusion protein comprising a hirudin amino acid sequence, synthesis, Figure 5 and its extension, Figure 5a, shows the construction of plasmid 35, pK370, which plasmid encodes a fusion protein comprising a hirudin amino acid sequence, and Figure 7 and its Figure 97939; 6a, show the synthesis of plasmid pKHIOI, which encodes a fusion of a monkey proinsulin amino acid sequence. oproteiinia.

The scale of the figures is generally not correct, above all the scale is "stretched" at the polylinker.

Example 1

By locating a Lac repressor [P.J. Farabaugh, Nature 274 (1978) 765-769] to plasmid pKK177-3 [Amann et al., Gene 25 (1983) 167] gives plasmid pJE118 (1) (Figure 10, cf. DE patent application P 35 26 995.2). , Example 6, Figure 6). This is opened at its single Aval restriction site and truncated in a manner known per se by exonuclease treatment of about 100 base pairs. After ligation, plasmid pEW100 (2) is obtained (Figure 1), which retains the entire Lac repressor gene but which, due to its smaller size, is clearly more abundant.

Instead of plasmid pKK177-3, the above-mentioned commercially available plasmid pKK223-3 can also be used as a starting product, a Lac repressor can be placed in it and the product obtained can be shortened accordingly.

Plasmid pEW10000 (2) is opened with the restriction enzymes EcoRI and SalI.

Plasmid (4) encoding hirudin, prepared according to Example 4 of DE-A-3 429 430 (EP-A-0 171 024) (Figure 3), is opened with the restriction enzymes Accl and SalI and a small fragment (5) containing most of it is isolated. hirudiinisek-sequence.

Plasmid p159 / 6 (8), prepared according to Example 4 of DE-A-3 419 995 (EP-A-0 163 249) (Figure 5), is opened with the restriction enzymes EcoRI and PvuI and a small fragment containing most of the IL is isolated. -2-sequence. This subsequence, and hereinafter also other truncated IL-2 sequences, is referred to in the figures as "IL2".

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Sequences (3), (5), (7) and the synthetic DNA sequence (8, Figure 1a) are then treated with T4 ligase. Plasmid pK360 (9) is obtained.

Competent E. coli cells are transformed with the ligase-5 thio product and inoculated onto NA plates containing 25 pg / ml ampicillin. Plasmid DNA of the clones is characterized by restriction and sequence analysis.

An overnight E. coli cell culture containing plasmid (9) is diluted in LB medium 10 (J.H. Miller, Experiments in Molecular Genetics, Cold

Spring Harbor Laboratory, 1972) containing 50 pg / ml ampicillin in a ratio of approximately 1: 100, and growth is monitored by measuring optical density (OD). With an OD of 0.5, the isopropyl-β-galactopyran-15 ranoside concentration (IPTG concentration) of the shake culture is adjusted to 1 mM, and the bacteria are separated by centrifugation after 150 to 180 minutes. The bacteria are boiled for 5 min in a buffer mixture (7 mol / l urea, 0.1% SDS, 0.1 mol / l sodium phosphate, pH 7.0), and the samples are transferred to an SDS gel electrophoresis plate. After electrophoresis, a protein band corresponding to the expected size of the fusion protein is obtained from the bacteria containing plasmid (9). After bacterial disruption (French Press, <R) Dyno-Muhle) and centrifugation, the fusion protein is present in the precipitate so that the supernatant can already be separated: significant amounts of other proteins. After isolation of the fusion protein, the desired hirudin peptide is released by bromocyanine cleavage. it is characterized after isolation by protein sequence analysis.

The induction conditions given apply to shake cultures, in the case of larger fermentations, correspondingly modified OD values and possibly slightly modified IPTG concentrations are appropriate.

Example 2

Plasmid (4) (Figure 1) is opened with Accl, and single-stranded ends are filled in with Klenow polymerase. It is then digested with SacI and the fragment (10) containing most of the hirudin sequence is isolated.

The commercially available vector pUC13 is opened with the restriction enzymes SacI and SmaI, and a large fragment is isolated (11).

Fragments (10) and (11) are then ligated into plasmid pK400 (12) (Figure 2) using T4 ligase. Plasmid (12) is shown in Figure 2 as two drawings, the lower 10 of which show the amino acid sequence of the hirudin derivative thus obtained.

Plasmid (4) (Figure 1) is opened with the restriction enzymes KpnI and SalI, and a small fragment (13) containing the hirudin subsequence is isolated.

Plasmid (12) is reacted with the restriction enzymes Hindi and KpnI, and a small fragment (14) containing the hirudin subsequence is isolated.

Plasmid (9) (Figure 1a) is partially digested with EcoRI, the free ends are filled in with Klenow polymerase in a 20-fill-in reaction, and digested with SalI. A derivative of plasmid pK360 is obtained (15).

Ligation of fragments (3), (13), (14) and (15) yields plasmid pK410 (16), shown twice in Figure 2a, the lower drawing showing the amino acid sequence of the fusion protein and thus the hirudin derivative obtained by acid cleavage.

After expression and treatment according to Example 1, a new hirudin derivative having the amino acids proline and histidine at positions 1 and 2 is obtained. This hiru-30 din derivative has similar activity to the natural product of DE, which has the amino acids threonine and tyrosine at these positions, but is more resistant to the action of aminopeptidases, which may be advantageous in in vivo use.

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Comparative Example 3

The commercially available vector pBR322 is opened with BamHI to give a linearized plasmid (17). The single-stranded ends are partially filled in using dATP, 5 dGTP, and dTTP, and the excess nucleotide G is removed with S1 nuclease to give the pBR322 derivative (18).

The HaellI fragment (19) of the monkey proinsulin gene [Wetekam et al., Gene 19 (1982) 181] is ligated with the modified plasmid (18) to give plasmid pPH1 (20). Because the insulin subsequence was inserted into the tetracycline gene, clones containing this plasmid are not tetracycline resistant and can thus be identified.

Plasmid (20) is opened with BamHI and DdeI, and a small fragment (21) is isolated.

In addition, the DdeI-PvuII subsequence is isolated from the monkey proinsulin sequence (22).

The vector pBR322 is opened with BamHI and PvuII, and the linearized plasmid is isolated (23).

Ligation of the insulin subsequences (21) and (22) with the opened plasmid (23) gives plasmid pPH5 (24). This is opened with BamHI and PvuII, and a small fragment is isolated (25).

To complete the insulin structure, a * 25 DNA sequence is synthesized (26).

The commercially available vector pUC8 is opened with BamHI and SalI, and the residual plasmid is isolated (27). This is ligated with the DNA sequences (25) and (26) to plasmid pPHi5 (28). This is opened with Säll, and filled with 30 single-stranded ends. From the obtained plasmid derivative (29), the DNA sequence (30) is cleaved with BamHI.

The commercially available vector pUC9 is opened with BamHI and SmaI, and a large fragment is isolated (31). This is ligated with the DNA sequence (30) to give plasmid pH16 (32).

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Plasmid1 (32) is opened with SalI, partially filled with linearized plasmid1 (33) with dCTP, dGTP and dTTP, and the remaining nucleotide is cleaved with T1I nuclease. The plasmid derivative (34) thus obtained is treated with BamHI, and an additional single strand of S1 nuclease is removed from the product (35) to give the plasmid derivative (36).

The blunt ends of the plasmid derivative (35) are cyclized to plasmid pPH 2 O (37).

Competent E. coli Hb 101 cells are transformed with the ligation mixture and seeded on a selective medium. Clones containing the desired plasmid produce proinsulin, and 28 of the 70 clones examined contained radioimmunologically detectable proinsulin. Plasmids are further characterized by DNA sequence analysis. They contain DNA with a codon encoding arginine above the codon corresponding to the first amino acid (Phe) of the B chain.

Plasmid (37) is digested with HindIII, filled into the internal ends of yk-20, and re-digested with DdeI. Isolate a small fragment (38).

Plasmid (28) (Figure 3a) is digested with SalI and DdeI, and a small fragment (39) is separated.

Plasmid (9) (Figure 1a) is first digested with Accl, filled in the free ends, and then digested with EcoRI.

A fragment (40) containing a truncated IL-2 sequence is isolated.

The linearized plasmid (3) (Figure 1) and DNA segments (38), (39), and (40) are then ligated into plasmid pPH30 (41). This plasmid encodes a fusion protein containing the following amino acid sequence after amino acids 1 to 114 of IL-2:

Asp-Phe-Met-Ile-Thr-Thr-Tyr-Ser-Leu-Ala-Ala-Gly-Arg.

12 97239 Arginine, the last amino acid in this intermediate Y, allows the insulin chain to be cleaved by trypsin.

Plasmid (9) (Figure 1a) can also be accessed to plasmid-5 (41) by the following route: (9) opening with Accl, filling in single-stranded ends, digestion with SalI, and ligating the resulting plasmid derivative (42) to segments (3), ( 38) and (39).

Example 4 Plasmid (6) (Figure 1) is opened with the restriction enzymes TaqI and EcoRI, and a small fragment (43) is isolated. This fragment is ligated to the synthesized DNA sequence

(44) and segments (3) and (5) to plasmid pPHIOO

(45). This plasmid encodes a fusion protein in which, after the first 15,132 amino acids of IL-2, there is an intermediate Asp-Pro followed by a hirudin amino acid sequence. Proteolytic cleavage thus yields a modified, biologically active IL-2 'with Asp at position 133 instead of Thr, and a hirudin derivative containing a group Pro at the N-terminus before the amino acid sequence of 20 natural products. This product is also biologically active and more resistant to proteases compared to the natural product.

The IL-2 'hirudin fusion protein is also biologically active: In a proliferation assay performed on an IL-2-dependent cell line (CTLL2), biological activity was observed.

After denaturation in 6 M guanidinium hydrochloride solution followed by renaturation in buffer solution (10 mM TrispHCl, pH 8.5, 10 mM EDTA), further high IL-2 activity was observed. In addition, the clotting time of acid-treated blood to which thrombin had been added was prolonged after the addition of the fusion protein.

The product was thus a bifunctional fusion protein.

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Example 5

The commercially available vector pUC12 is opened with the restriction enzymes EcoRI and SacI. An IL-2 subsequence is inserted into this linearized plasmid (46), which is loh-5 banded from plasmid (6) (Figure 1) with the restriction enzymes EcoRI and SacI. This sequence (47) contains complete triplets corresponding to the first 94 amino acids of IL-2. Ligation (46) and (47) yields plasmid pK300 (48).

Plasmid (9) (Figure 1a) is opened with EcoRI, filled into single-stranded ends, and then digested with HindIII.

A small fragment (49) containing a portion of the pUC12 polylinker after the DNA segment encoding hirudin is isolated.

Plasmid (48) is opened with restriction enzymes SmaI 15 and HinII, and a large fragment (50) is isolated. Ligation to (50) (49) yields plasmid pK301 (51).

Competent E. coli 294 cells are transformed with the ligation mixture. Clones containing plasmid (51) are characterized by restriction analysis. They contain 20 DNAs with codons corresponding to a 6-amino acid linking sequence after the codons corresponding to the first 96 amino acids of IL-2, followed by codons corresponding to hirudin.

Plasmid (51) is treated with EcoRI and HindIII, and a fragment (52) containing the DNA sequence corresponding to said eukaryotic fusion protein is isolated.

Plasmid (2) (Figure 1) is opened with EcoRI and Hind III. The resulting linearized plasmid (53) is ligated with the DNA sequence (52) to give plasmid pH370 (54).

When plasmid (54) is expressed in E. coli in a manner similar to Example 1, a fusion protein with an intermediate of 14 97239 after the first 96 amino acids of IL-2 is obtained.

Ala-Gln-Phe-Met-Ile-Thr followed by the amino acid sequence of hirudin.

Comparative Example 6 From the plasmid (41) (Example 3; Figure 3c), the DNA segment encoding monkey proinsulin was cleaved with restriction enzymes EcoRI and HindIII, and the single-stranded ends were filled in. A DNA segment (55) is obtained.

Plasmid (48) (Example 5, Figure 5) is opened with SmaI-10 and treated with alkaline bovine phosphatase. The linearized plasmid (56) thus obtained is ligated with the DNA segment (55) to give plasmid pK302 (57). E. coli 294 cells are transformed with the ligation mixture, and clones containing the desired plasmid are characterized by subjecting the plasmid DNA first to restriction and then to sequence analysis.

From plasmid (57), a segment (58) encoding IL-2 and monkey proinsulin is cleaved with EcoRI and HindIII.

Plasmid (2) (Example 1, Figure 1) is similarly digested with EcoRI and HindIII, and segment (58) is ligated to the linearized plasmid (3). Plasmid pKHIOI (59) is obtained.

Expression according to Example 1 results in a fusion protein in which the first 96 amino acids of IL-2 are followed by a 14 amino acid fusion sequence (corresponding to sequence Y in the DNA segment (58)) followed by the amino acid sequence of monkey proinsulin.

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Annex I: DNA sequence of interleukin-2

Triplet No. 012 5 Amino Acid Ala Pro

Nucleotide No. 1 10

Coding thread 5 'AA TTC ATG GCG CCG

Uncoded thread_2J_G TAC CGC GGC_ 3 4 5 6 7 8 9 10 11 12 10 Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu

20 30 40 ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG

TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC

13 14 15 16 17 18 19 20 21 22

Gin Leu Glu His Leu Leu Leu Asp Leu Gin 15 50 60 70

CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG

GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC

23 24 25 26 27 28 29 30 31 32

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 ^ 80 90 100

ATG ATC CTG AAC GGT ATC AAC. AAC TAC AAA

TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT 1 34 35 36 37 38 39 40 41 42

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe 25 110 120 130

AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC

TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG

16 97239 43 44 45 46 47 48 49 50 51 52

Lys Phe Tyr Met Pro Lys Lye Ala Thr Glu 140 150 160

AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA

5 TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT

53 54 55 56 57 58 59 60 61 62

Leu Lye His Leu Gin Cys Leu Glu Glu Glu 170 180 190

CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG

10 GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC

63 64 65 66 67 68 69 70 71 72

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu 200 210 220

CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG

15 GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC

73 74 75 76 77 78 79 80 81 82

Ala Gin Ser Lys Asn Phe His Leu Arg Pro 230 240 250

GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG

20 CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC

83 84 85 86 87 88 89 90 91 92

Arg Asp Leu lie Ser Aen lie Aen Val lie 260 270 280

CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC

25 GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG 1: 8 tt-l ill) In # :: 94 95 96 97 98 99 100 101 102

Val Leu Glu Leu Lye Gly Ser Glu Thr Thr 290 300 310

GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG

30 CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC

17 97239 103 104 105 106 107 108 109 110 111 112

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 320 330 340

TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG

5 AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC

113 114 115 116 117 118 119 120 121 122

Thr Ile Val Glu Phe Leu Asn Arg Trp lie 350 360 370

ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC

10 TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG

123 124 125 126 127 128 129 130 131 132

Thr Phe Cys Gin Ser lie lie Ser Thr Leu 380 390 400

ACC TTC TGC CAG TCG ATc 'ATC TCT ACC CTG

15 TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC

133 134 135

Thr 410 ACC TGA TAG 3 '2Q TGG ACT ATC AGC T 5' • ·

Claims (7)

A method of producing a fusion protein, characterized in that a gene is expressed in a bacterial host cell, which gene encodes a C or N-terminal moiety that substantially corresponds to the amino acid sequence of interleukin-2 and further encodes hirudin. The method of claim 1, characterized in that the gene encodes a fusion protein of the general formula Met - X - Y - Z (Ia) or Met - Z - Y - X (Ib) in which formulas X substantially correspond to the amino acid sequence of human interleukin-2, Y is a direct link or a bridge moiety formed of genetically encodable amino acids and Z is the amino acid sequence of hirudin. 3. A process according to claim 2, characterized in that Y next to Z contains the amino acid Met, Cys, Trp, Arg or Lys or consists of these amino acids. Method according to claim 2, characterized in that Y next to Z contains the amino acid sequence Asp - Pro or consists thereof. Process according to any one of claims 1-4, characterized in that the fusion protein is separated from soluble proteins by centrifugation. Method according to any one of claims 1-5, characterized in that the host cell is E. coli. 7. Plasmid pPH101, presented in Figure 4.