AU595262B2

AU595262B2 - Eukaryotic fusion proteins, the preparation and use thereof and means for carrying out the process

Info

Publication number: AU595262B2
Application number: AU65693/86A
Authority: AU
Inventors: Paul Habermann; Friedrich Wengenmayer
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1985-11-27
Filing date: 1986-11-26
Publication date: 1990-03-29
Anticipated expiration: 2006-11-26
Also published as: ZA868943B; FI864798A; DK52292D0; EP0468539B1; EP0227938A3; IL80755A0; IE863119L; FI864798A0; DK568586A; EP0468539A1; JP2566933B2; DE3684892D1; EP0227938A2; FI93471B; ES2077747T3; FI93471C; ATE127841T1; PT83813B; DE3650396D1; NO176481B

Abstract

Fusion proteins in which the C- or N-terminal essentially corresponds to the first 100 units of interleukin 2 are new. Also new are gene structures coding for the above fusion proteins, vectors containing these gene structures, and hot cells containing these vectors. Hirudin derivs. with an amino acide sequence begining N-terminally with Pro are new and claimed. Human interleukin 2 derivs. contg. Asp. C-terminally are new and claimed.

Description

Form COMMONWEALTH OF AIJSTRALIA PATENTS ACT 1952-69 'COMPLETE SPECIFICATION

(DRIGINAL)

Glass Int. Class Application Number: Lodged: 6~6~3/~6.

Complete Specification Lodged: Accepted: Puiblish-ed: *Jriority: Related Art A5526 [This C tconttills Lh sl' al-:d .cra'L t' a S Name of Applicant: HOECHST AKTI ENGESELLSCHAFT Atidress of Applicant: .o~tual Inventor: ','Jdress for Service: Bruningstrasse, D-6230 Frankfurt/Main 80, Federal Republic of Germany PAUL HABERMANN and FRIEDRICH WENGENMAYER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the Invention entitled: EUL(ARYOTIC FUSION PROTEINS, THE PREPARATION AND USE THEREOF AND MEANS FOR CARRYING OUT TH4E PROCESS The following statement is a full description of this invention, including the best met~hod of performing it known to'-u I1.

Hoechst t, A ltngeaellechaft PAT 510 p' $~At Ieht F Pr kurist Authorized signat ppa. Reuter i.V. Lapice -la- EUKARYOTIC FUSION PROTEINS, THE PREPARATION AND USE THEREOF, AND MEANS FOR CARRYING OUT THE PROCESS The invention relates to an "open reading frame" from a DNA which codes for interleukin-2, and to the use of this DNA as an aid for the expression of peptides and proteins.

In the preparation of eukaryotic proteins by genetic engineering, the yield obtained in bacteria is frequently only low, especially in the case of small proteins which have a molecular weight up to about 15,000 Daltons and whose structures contain disulfide bridges. It is asssumed that the proteins which have been produced are rapdily degraded by proteases intrinsic to the host. For this reason, it is expedient to construct gene structures which code for fusion proteins, the undesired section of the fusion protein being a protein which is intrinsic to the host and which, after isolation of the primary product, is e cleaved off by methods known per se. Fusion proteins thus so 0 act as an intermediate product from which the desired eo.. 20 product is set free by known chemical or enzymatic S"processes. The fused protein is not recognised by the &So bacterial host cell az a "foreign" protein and thus not degraded by the bacterial host. As a further precaution genes for fusion proteins 4enerally are designed in such a 00 25 manner that the expressed fusion protein is insoluble which 2 p des a further protection against the proteases 0*00" providsafrhrpoeto agis th potss intrinsic to the host. It has now been found, surprisingly, that an N-terminal section of interleukin-2 which essentially corresponds to the first 100 amino acids is especially well suited as the "ballast" in the preparation of fusion proteins. Thus, the primary product obtained is a a a 0 fusion protein which is composed entirely or very predominantly of eukaryotic protein sequences.

Surprisingly, this eukaryotic protein can serve as the ballast moiety of such fusion proteins and is apparently not recognized as foreign protein in the relevant host organism, -Ibnor is it immediately degraded again. Another advantage is that the fusion proteins according to the invention are sparingly soluble or insoluble and thus can straightforwardly be removed from the soluble proteins, expediently by centrifugation.

Since it is unimportant accordingly to the invention for the function of the fusion protein as ballast section whether the interleukin-2 section represents a biologically active molecule, nor is the exact structure of the interleukin-2 S 0 S 3 A.1 2 section of importance either. For this purpose it is sufficient that essentially the first 100 N-terminal amino acids are present. Thus, it is possible, for example, to undertake at the N-terminal end modifications which allow cleavage of the fusion protein if the desired protein is located N-terminal thereto. Conversely, it is possible to undertake C-terminal modifications in order to make it possible or easier to cleave off the desired protein if as customary the latter is C-terminal bonded in the fusion protein.

The natural DNA sequence coding for human interleukin-2, "IL-2" in the text which follows, is known from the European Patent Application with the Publication No. EP-Al- 0,091,539. The Literature cited there also relates to 15 IL-2 from mice and rats. This mammalian DNA can be used for the synthesis of the proteins according to the invention. However, it is more expedient to start from a synthetic DNA, and especially advantageously from the DNA for human IL-2 which has been proposed in the (non-prior- 20 published) German Offenlegungsschrift 3,419,995 (corresponding to the European Patent Application published under the No. 0,163,249). This synthetic DNA sequence is depicted in the appendix (DNA sequence This synthetic DNA not only has the advantage that its choice of codons 25 is suited to the circumstances in the host which is used '.most often, E. coli, but it also contains a number of cleavage sites for restriction endonucleases which can be utilized according to the invention. Table 1 which follows gives a selection of the suitable cleavage sites at the start and in the region of the 100th triplet.

However, this does not rule out the possibility of undertaking modifications in DNA in the intermediate region, it being possible to make use of the other cleavage sites listed in the abovementioned patent application.

3 -3- TABLE 1 Restriction Recognition Position of the first enzyme sequence nucleotide of the recognition sequence (coding strand) 3' Aha II, Ban I, Hae II, Nar I, GGCGCC 8 Ban II, Sac I, 10 Sst I GAGCTC 291 Hha I GCGC 9 Hinf I GACTC Pvu I CGATCG 346 Taq I TCGA 387 15 If use is made of the nucleases Ban II, Sac I or Sst I then an IL-2 part-sequence which codes for about 95 amino acids is obtained. This length is generally sufficient to obtain an insoluble fusion protein. If the solubiLity is still insufficiently low, for example in the case of 20 a desired hydrophilic eukaryotic protein, but it is not intended to make use of the cleavage sites Located nearer to the C-terminal end in order to produce as Little ballast" as possibLe then it is possible to extend the DNA sequence at the N- and/or C-terminal end, by appropriate adaptors or linkers, and thus "tailor" the "ballast" section. Of course, it is also possible to use the DNA J sequence more or Less right up to the end and thus generate IL-2 which is biologically active, and optionally modified, as a "byproduct" or generate a bifunctional protein which has the action of IL-2 in addition to the action of the coded protein.

Thus the invention relates to fusion proteins of the general formula |i 4 Met X Y Z or Met Z Y- X (la) (Ib) in which X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, preferably, human IL-2, Y denotes a direct bond if the amino acid or amino acid sequence adjacent to the desired protein allows the desired protein to be cleaved off, or otherwise denotes a bridging element which is composed of one or more S 10 genetically codable amino acids and permits the cleavage Soff, and Z is a sequence of genetically codable amino acids representing the desired protein.

1 As is evident from the formulae Ia and Ib and as has already been mentioned above it is possible to bring about the expression of the desired protein upstream or downstream of the IL-2 section. For simplicity, in the following text essentially the first option, which corresponds to the conventional method for the preparation 20 of fusion proteins, will be illustrated. Thus, although this "classic" variant is described below, this is not intended to rule out the other alternative.

The fusion protein can be cleaved chemically or enzymatically in a manner known per se. The choice of the suitable method depends in particular on the amino acid sequence of the desired protein. For example, if the latter contains no methionine, Y can denote Met and then chemical cleavage with cyanogen chloride or bromide is carried out.

If there is cysteine at the carboxyl terminal end of the linking element Y, or if Y represents Cys, then an enzymatic cysteine-specific cleavage or a chemical cleavage, for example after specific S-cyanylation, can be carried out. If there is tryptophan at the carboxyl terminal end of the bridging element Y, or if Y represents Trp, then chemical cleavage with N-bromosuccinimide can be carried out.

5 Proteins which do not contain Asp Pro in their amino acid sequence and are sufficiently stable to acid can be cleaved proteolytically in a manner known per se.

This results in proteins which contain N-terminal proline and C-terminal aspartic acid respectiveLy. It is thus also possible in this way to synthesize modified proteins.

The Asp-Pro bond can be made more labile to acid if this bridging element is (Asp)n-Pro or Glu-(Asp),-Pro, n denoting 1 to 3.

9* Examples of enz)matic cleavages are Likewise known, it also being possible to make use of modified enzymes with S improved specificity (cf. C.S. Craik et at., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, then it is expedient to choose as sequence Y a peptide sequence in which an amino acid which can be *cleaved off with trypsin (Arg, Lys) is bonded to the Nterminal amino acid (Phe) of proinsulin, for example Ala-Ser-Met-Thr-Arg, since it is then possible to carry 20 out the arginine-specific cleavage with the protease trypsin.

If the desired protein does not contain the amino acid sequence ILe-Glu-Gly-Arg, then the fusion protein can be cleaved with factor Xa (European Patent Applications with the Publication Nos.

0,025,190 and 0,161,973).

The fusion protein is obtained by expression in a suitable expression system in a manner known per se. Suitable for this purpose are all known host vector systems, that is to say, for example, mammalian cells and microorganisms, for example yeasts and, preferably, bacteria, in particular 6- E. coli.

The DNA sequence which codes for the desired protein is incorporated in a known manner in a vector which ensures satisfactory expression in the selected expression system.

In bacterial hosts it is expedient to choose the promotor and operator from the group Lac, tac, trp, PL or PR of phage X, hsp, omp or a synthetic promotor as proposed in, for example, German Offenlegungsschrift 3,430,683 (European Patent kpplication with the Publication No. 0,173,149).

10 The tac promotor-operator sequence is advantageous and is now commercially available (for example expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63).

r It may prove expedient in the expression of the fusion protein according to the invention to modify some of the triplets for the first few amino acids downstream of the ATG start codon in order to prevent any base-pairing at the mRNA level. Modifications of this type, as well as modifications, deletions or additions of individual amino acids 00 20 in the IL-2 protein section, are familiar to the expert and the invention likewise relates to them.

The invention is illustrated in detail in the examples which follow and in the figures. In these, Figure 1, and its continuation Figure la, relate to the synthesis of the plasmid pK360 which codes for a fusion protein which has the hirudin sequence; Figure 2, and its continuation Figure 2a, relate to the synthesis of the plasmid pK410 which Likewise codes for a fusion protein having the amino acid sequence of hirudin, Figure 3, and its continuations Figures 3a to 3c, relate to the construction of the plasmids pPH15, 16, 20 and which code for fusion proteins which contain the amino acid 7 sequence of monkey proinsuLin, Figure 4 relates to the synthesis of the pLasmid pPH100 which codes for a fusion protein having the amino acid sequence of hirudin, Figure 5, and its continuation Figure 5a, relate to the construction of the plasmid pK370 which codes for a fusion protein having the amino acid sequence of hirudin, and Figure 6, and its continuation Figure 6a, relate to the synthesis of the plasmid pKH101 which codes for a fusion protein having the amino acid sequence of monkey proinsuLin.

In general, the figures are not drawn to scaLe; in particular, the scale has been "stretched" in depicting the poLyLinkers.

0 0* 0 0*00 0@ 0 000 EXAMPLE 1 15 The plasmid pJF118 is obtained by insertion of the Lac repressor Farabaugh, Nature 274 (1978) 765-769) into the plasmid pKK 177-3 (Amann et aL., Gene 25 (1983) 167) (Fig. 1; cf. German Patent Application P 35 26 995.2, ExampLe 6, Fig. pJF118 is opened at the unique 20 restriction site for Ava I and is shortened by about 1,000 bp in a manner known per se by exonucLease treatment.

Ligation resuLts in the plasmid pEW 1000 (Figure 1) in which the Lac repressor gene is fuLLy retained but which, by reasor, of the shortening, is present in a distinctLy higher copy number than the starting plasmid.

In place of the pLasmid pKK177-3, it is also possible to start from the abovementioned commercially available pLasmid pKK223-3, to incorporate the Lac repressor, and to shorten the resulting product analogously.

The plasmid pEW 1000 is opened with the restriction enzymes EcoR I and Sal I id-i*CP -8 The plasmid which codes for hirudin and has been prepared as in German Offenlegungsschrift 3,429,430 (European Patent Application with the Publication No. 0,171,024), Example 4 (Figure is opened with the restriction enzymes Acc I and Sal I, and the small fragment which mostly contains the hirudin sequence is isolated.

The plasmid p 15 9 6 prepared as in German Offenlegungsschrift 3,419,995 (European Patent Application with the Publication No. 0,163,249), Example 4 (Figure is opened with the restriction enzymes Eco RI and Pvu I, and the small fragment which contains most of the IL-2 sequence is isolated. This part-sequence and other shortened IL-2 sequences in the text which follows are identified by "AIL2" in the figures.

0* 15 Thereafter the sequences and the synthetic DNA sequence Figure la) are treated with T4 Ligase.

The plasmid pK360 is obtained.

Competent E. coli ceLLs are transformed with the ligation product and plated out on NA plates which contain 25 pg/mL 20 ampicillin. The plasmid DNA of the clones is characterized by restriction and sequence analysis.

An overnight culture of E. coli cells which contain the plasmid is diluted in the ratio of approximately 1:100 with LB medium Miller. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) which contains 50 pg/ml ampicillin, and the growth is monitored by measurement of the OD. When the OD is 0.5, the shake culture is adjusted to 1 mM isopropyl B-galactopyranoside (IPTG) and, after 150 to 180 minutes, the bacteria are spun down. The bacteria are boiled in a buffer mixture (7M urea, 0.1% SDS, 0.1 M sodium phosphate, pH for 5 minutes, and samples are applied to a SDS gel electrophoresis plate. Bacteria which contain the plasmid (9) provide after electrophoresis a protein band which corresponds to the size of the expected fusion protein.

-9- Disruption of the bacteria (French Press; (R)Dyno mill) and centrifugation results in the fusion protein being located in the sediment so that considerable amounts of the other proteins can now be removed with the supernatant.

After isolation of the fusion protein, cleavage with cyanogen bromide results in liberation of the expected hirudin peptide. The latter is characterized after isolation by protein sequence analysis.

The indicated induction conditions apply to shake cultures; with larger fermentations appropriately modified OD values and, where appropriate, slight changes in the IPTG concentrations are expedient.

EXAMPLE 2 The plasmid (Figure 1) is opened with Acc I, and the protruding ends are filled in with Klenow polymerase.

Then cleavage with Sac I is carried out, and the fragment (10) which contains most of the hirudin sequence is iso-

S.

ated.

The commercially available vector pUC 13 is opened with 20 the restriction enzymes Sac I and Sma I, and the large fragment (11) is isolated.

Using T4 ligase, the fragments (10) and (11) are now ligated to give the plasmid pK 400 (12) (Fig. The plasmid (12) is shown twice in Figure 2, the lower representation emphasizing the amino acid sequence of the hirudin derivative which can thus be obtained.

The plasmid (Figure 1) is opened with the restriction enzymes Kpn I and Sal I, and the small fragment (13) which contains the hirudin part-sequence is isolated.

The plasmid (12) is reacted with the restriction enzymes Hinc II and Kpn I, and the small fragment (14) which contains the hirudin part-sequence is isolated.

i I; _1~1_ 10 The plasmid (Figure la) is partially cleaved with EcoR I, the free ends are subjected to a fill-in reaction with KLenow polymerase, and Sal I cleavage is carried out.

The derivative (15) of the plasmid pK360 is obtained.

Ligation of the fragments (14) and (15) results in the plasmid pK410 (16) which is shown twice in Figure 2a, the lower representation showing the amino acid sequence of the fusion protein and thus that of the hirudin derivative obtained after acid cleavage.

10 Expression and working up as in Example 1 results in a new hirudin derivative which has the amino acids proline and histidine in positions 1 and 2. This hirudin derivative has the same activity as the natural product, according to German Offenlegungsschrift 3,429,430, which has the 15 amino acids threonine and tyrosine in these positions, but is more stable to attack by aminopeptidases, which may result in advantages for in vivo use.

EXAMPLE 3 The commercially available vector pBR 322 is opened with 20 Bam H I, this resulting in the linearized plasmid (17).

The free ends are partially filled in by use of dATP, dGTP and dTTP, and the protruding nucleotide G is split off with S1 nuclease, this resulting in the pBR 322 derivative (18).

The Hae III fragment (19) from monkey proinsulin (Wetekam et al., Gene 19 (1982) 181) is ligated with the modified plasmid this resulting in the plasmid pPH 1 Since the insulin part-sequence has been inserted into the tetracycline resistance gene, the clones which contain this plasmid are not resistant to tetracycline and thus can be identified.

The plasmid (20) is opened with Bam HI and Dde I, and the small fragment (21) is isolated.

MIN" j X essentially denotes the amino acid sequence of approximately the first 100 amino acids of human interleukin-2, Y denotes a direct bond or a bridging element which is composed of genetically codable amino acids and which i i 11 In addition, the Dde I-Pvu II part-sequence (22) from the monkey proinsulin sequence is isolated.

The vector pBR 322 is opened with Bam HI and Pvu II, and the Linearized plasmid (23) is isolated,, Ligation of the insulin part-sequences (21) and (22) with the opened plasmid (23) results plasmid pPH5 (24).

The Latter is opened with Bam HI and Pvu II, and the small fragment (25) is isolated.

The DNA sequence (26) to make up the insulin structure 10 is synthesized.

The commercially available vector pUC 8 is opened with the enzymes Bam HI and Sal I, and the remainder of the plasmid (27) is isolated. The latter is ligated with the DNA sequences (25) and (26) to give the plasmid pPH 15 (28).

15 The latter is opened with Sal I and the protruding ends are filled in. Bam HI is used to cleave the DNA sequence 9.

(30) off the resulting plasmid derivative (29).

The commercially available vector pUC 9 is opened with the enzymes Bam HI an Sma I, and the large fragment (31) is 20 isolated. The latter is ligated with the DNA sequence this resulting in the plasmid pPH16 (32).

The plasmid (32) is opened with Sal I, and the Linearized plasmid (33) is partially filled in with dCTP, dGTP and dTTP, and the remaining nucleotide T is cleaved off with S1 nuclease. The resulting plasmid derivative (34) is treated with Bam HI, and the protruding single strand is removed from the product (35) with S1 nuclease, this resulting in the plasrid derivative (36).

The blunt ends of the plasmid derivative (35) are cyclized to give the plasmid pPH 20 (37).

Competent E. coli Hb 101 cells are transformed with the 12 ligation mixture and plated out on selective medium. Clones which contain the desired plasmid express proinsulin, and 28 of 70 clones tested radioimmunologically contained detectable proinsulin. The plasmids are also characterized by DNA sequence analysis. They contain DNA which codes for arginine upstream of the codon for the first amino acid of the B chain (Phe).

The plasmid (37) is cleaved with Hind III, the protruding ends are filled in, and then cleavage with Dde I is carried out. The small fragment (38) is isolated.

The plasmid (28) (Figure 3a) is cleaved with Sal I and Dde I, and the small fragment (39) is separated off.

The plasmid (Figure la) is initially cleaved with Acc I, the free ends are filled in, and then partial cleavage with Eco RI is carried out. The fragment (40) which cont, ains the shortened IL-2 sequenee is isolated.

L' The linearized plasmid (Figure 1) and the DNA segments (39) and (40) are now ligated to give the plasmid 'pPH 30 This plasmid codes for a fusion protein which 20 has, downstream of amino acids 1 to 114 of IL-2, the following amino acid sequence: 't Asp-Phe-Met-Ile-Thr-Thr-Tyr-Ser-Leu-Ala-ALa-Gly-Arg.

i The arginine which is the last amino acid in this bridging element Y makes it possible to cleave off the insulin chains with trypsin.

It is also possible starting from plasmid (Figure la) to obtain plasmid (41) by the following route: is opened with Acc I, the protruding ends are filled in, then cleavage with Sal I is carried out, and the resulting plasmid derivative (42) is Ligated with the segments (38) and (39).

13 EXAMPLE 4 The plasmid (Figure 1) is opened with the restriction enzymes Taq I and Eco RI, and the small fragment (43) is isolated. This fragment is ligated with the synthesized DNA sequence (44) and the segments and to give the plasmid pPH 100 This plasmid codes for a fusion protein in which the first 132 amino acids of IL-2 are followed by the bridging element Asp-Pro and then by the amino acid sequence of hirudin. Thus proteolytic cleavage provides a modified, biologically active IL-2' which contains Asp in place of Thr in position 133, and a hirudin Sderivative which contains an N-terminal Pro upstream of the amino acid sequence of the natural product. This product o* is also biologically active and, compared with the natural 15 product, is more stable to attack by proteases.

S The IL-2' hirudin fusion protein also has biological activity: Biological activity was found in a cell proliferation test using an IL-2-dependent cell line (CTLL2).

20 Furthermore, after denaturation in 6 M guanidinium hydrochloride solution followed by renaturation in buffer solution (10 mM tris-HCl, pH 8.5, 1 mM EDTA), high IL-2 activity was found. In addition, the coagulation time of acidtreated blood to which thrombin had been added was increased after addition of the fusion protein.

Thus a bifunctional fusion protein is obtained.

EXAMPLE The commercially available vector pUC 12 is opened with the restriction enzymes Eco RI and Sac I. Into this linearized plasmid (46) is inserted an IL-2 part-sequence which has been cleaved out of the plasmid (Figure 1) with the restriction enzymes Eco RI and Sac I. This sequence (47) comprises the complete triplets for the first 94 14 amino acids of IL-2. Ligation of (46) and (47) results in the plasmid pK 300 (48).

The plasmid (Figure la) is opened with Eco RI, the protruding ends are filled in, and then cleavage with Hind III is carried out. The small fragment (49) which contains part of the polylinker from pUC 12 downstream of the DNA sequence coding for hirudin is isolated.

The plasmid (48) is opened with the restriction enzymes Sma I and Hind III, and the large fragment (50) is iso- Lated. Ligation of (50) with (49) results in the plasmid pK 301 (51).

The ligation mixture is used to transform competent E.

coli 294 cells. Clones which contain the plasmid (51) are characterized by restriction analysis. They contain DNA 15 in which the codons for the first 96 amino acids of IL-2 are followed by codons for a bridging element of 6 amino acids and, thereafter, the codons for hirudin.

m S. The plasmid (51) is reacted with Eco RI and Hind III, and the fragment (52) which contains the DNA sequence for the 20 said eukaryotic fusion protein is isolated.

"The plasmid (Figure 1) is opened with Eco RI and Hind III. The resulting linearized plasmid (53) is ligated with the DNA sequence this resulting in the plasmid pK 370 (54).

When expression of the plasmid (C4) is effected in E. coli as in Example 1, the fusion protein obtained has the first 96 amino acids of IL-2 followed by the bridging element Ala-Gln-Phe-Met-Ile-Thr and, thereafter, the amino acid sequence of hirudin.

EXAMPLE 6 Using the restriction enzymes Eco RI and Hind III, the DNA segment which codes for monkey proinsuLin is cleaved out of the plasmid (41) (Example 3; Figure 3c), and the protruding ends are fiLLed in. The DNA segment (55) is obtained.

The pLasmid (48) (Example 5, Figure 5) is opened with Sma I and treated with bovine alkaline phosphatase. The resulting linearized plasmid (56) is ligated with the DNA segment this resulting in the plasmid pK 302 (57).

E. coli 294 cells are transformed with the ligation mixture, and clones containing the desired plasmid are characterized first by restriction analysis and then by sequence anaLysis of the plasmid DNA.

S.

m 15 Using Eco RI and Hind III, the segment (58) which codes for IL-2 and monkey proinsulin is cleaved out of the plasmid (57).

The plasmid (Example 1, Figure 1) is likewise cleaved with Eco RI and Hind III, and the segment (58) is Ligated 20 into the linearized plasmid The plasmid pKH 101 (59) is obtained.

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

I

APPENDIX I: DNA sequence of interLeukin-2 Triplet No.

Amino acid Nucleotide No.

Cod. strand 0 1 2 Met Ala Pro ATG GOG COG 5 AA TTC 0 @009 0 @0 0 0O 000* *0 0 0 000 e.

S S *50* 0e S S

S*

S

S.

*5*5*5 *5*55*

S

5050 55 0 0 *5

I

Non-.cod, strand 31 G TAO OGO GGC 3 4 5 6 7 8 9 10 11 12 Thr Ser Ser Ser Thr Lys Lys Thr Gin Leu 30 ACC TCT TOT TOT ACC AAA AAG ACT CAA OTG TGG AGA AGA AGA TGG TTT TTO TGA GTT GAO 13 14 15 16 17 18 19 20 21 22 Gin Leu Giu His Leu Lieu Leu Asp Leu Gin 50 60 CAA CTG GAA CAC CTG OTG OTG GAO CTG CAG GTT GAO OTT GTG GAO GAO GAO CTG GAC GTC 23 24 25 26 27 28 29 30 31 32 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 90 100 ATG ATO CTG AAC GGT ATO AAC AAC TAO AAA TAO TAG GAO TTG OCA TAG TTG TTG ATG TTT 33 34 35 36 37 38 39 40 41 42 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe 110 10130 AAC COG AAA OTG AOG COT ATG OTG ACC TTC TTG GO TTT GAO TGC GCA TAO GAO TOG AAG 17 43 Lys

AAA

44 Ph e 140

TTC

45 46 47 48 Tyr Met Pro Lys 150 TAO ATG COG AAA 49 Lys

AAA

TTT

50 51 Ala Thr 160 GOT ACC OGA TGG 52 Glu

GAA

OTT

00 00 0 0 0 000.

0 0 00 0 00 0 0 0000 00 0 0 000 0 00 0 0 0000 00 0 0000 00 0 0 00 000000 0 000000 0 00 0 0 00 0 00 00 53 54 55 56 57 58 59 60 61 62 Leu Lys His Leu Gin Cys Leu Giu Glu Glu 170 180 190 CTG AAA CAC OTO OAG TGT OTA GAA GAA GAG GAO TTT GTG GAG GTO ACA GAT OTT OTT OTO 63 64 65 66 67 68 69 70 71 72 Leu Lys Pro ILeu Giu Glu Val Leu Asn ILeu 200 210 220 OTG AAA OOG OTG GAG GAA GTT OTG AAO OTG GAO TTT GGO GAO OTO OTT CAA GAO TTG GAO 73 74 75 76 77 78 79 80 81 82 Ala Gin Ser Lys Asn Phe His Leu Arg Pro 230 240 250 GOT OAG TOT AAA AAT TTO CAC OTG OGT OOG OGA GTO AGA TTT TTA AAG GTG GAO GOA GGO 83 84 85 86 87 88 89 90 91 92 Arg Asp Leu Ile Ser Asn Ile Asn Va~l Ile 260 270 280 OGT GAO OTG ATO TOT AAO ATO AAO GTT ATO GOA CTG GAO TAG AGA TTG TAG TTG CAA TAG 93 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 OTO AAA GGT TOT GAA ACC AOG CAA GAO CTO GAG TTT COA AGA OTT TGG TGO 18 103 104 Phe M~et 320 TTC ATG AAG TAC 105 Cy s

TGC

ACG

106 107 108 Giu Tyr Ala 330 GAA TAO GCG CTT ATG OGO 109 110 Ill Asp Giu Thr 340 GAC GAA ACT CTG OTT TGA 112 Ala

GCG

CGC

113 Thr

ACG

99 TGC 123 Thr

.:~ACC

T GG 133 999999 Thr

ACC

*:Goo:

TGG

114 Ile 350 AT C

TAG

124 Ph e 380

TTC

AAG

115 116 117 118 Val Giu Phe Leu 360 GTT GAA TTT CTG CAA OTT AAA GAC 119 Asn

AAC

TTG

129 Ile

ATC

TAG

120 121 Arg Trp 370 OGT TGG GOA ACC 130 131 Ser Thr 400 TOT ACC AGA TGG 1 22 Ile

ATO

TAG

132 Leu

OTG

GAO

125 Cys

TGO

A OG 126 127 128 Gin Ser Ile 390 CAG TOG ATO GTO AGO TAG 134 135 410

TGA

ACT

TAG

AT 0 AGO T 9 99 99 9 .999 99 9 9 99 9 9.

Claims

1. A fusion protein which has a C- or N-terminal sec- tion which essentially corresponds to the first 100 amino acids of interleukin-2.

2. A fusion protein of the formula Met X Y Z or Met Z Y X (la) (Ib) in which X essentially denotes the amino acid sequence of approxi- mateLy the first 100 amino acids of human interLeukin-2, Y denotes a direct bond or a bridging element which is composed of genetically codable amino acids and which allows the amino acid sequence to be cleaved off and Z is a sequence of genetically codable amino acids.

3. A fusion protein as claimed in claim 2, wherein adjacent ro Z, contains Met, Cys, Trp, Arg or Lys, or consists of these amino acids. *feeo*

4. A fusion protein as claimed in claim 2, wherein Y, adjacent to Z, contains the amino acid sequence "Asp Pro, or consists of this sequence. A fusion protein as claimed in one or more of the preceding claims, wherein Z denotes the amino acid sequence of a proinsulin or of a hirudin.

6. A process for the preparation of a fusion protein as claimed in claims 1 to 5, which comprises expression of a gene structure coding for this protein in a host cell, and removal of the fusion protein. The plasmid pEW 1000 is opened with the restriction enzymes EcoR I and Sal I I&4 4 A i:ll~i- -i ~l -i~l-^-...~lilirri~ S S o 5 .4 OL 4 *55 is S 5 -5 4 *9 I 55 S S is 20

7. The process as claimed in claim 6, wherein the fusion protein is removed from the soluble proteins by centrifugation.

8. The process as claimed in claim 6 or 7, wherein the host cell is a bacterium.

9. The process as claimed in claim 8, wherein the host cell is E. coli. The use of the fusion protein as claimed in claims 1 to 5, or of the fusion proteins obtained as claimed in claims 6 to 9, for the preparation of the protein which essentially corresponds to the amino acid sequence Z'by chemical or enzymatic cleavage.

11. A gene structure coding for a fusion protein as claimed in claims 1 to

12. A vector containing a gene structure as claimed in claim 11.

13. Plasmids pEW 1000, pK360, pK410, pPH30, pPH100, Pk370 and pKH101 cs ere\ \ore

14. A host cell containing a vector as claimed in claim 12. A h irudint-e-p ivat wive sh ie has oC sequence which starts with an N-terminal Pro

16. A hirudin der" ive as claimed in claim 15 whose amino acid-s-equence starts with N-terminal Pro-His or Pro- -T1 l I Asp. A human IL-2 derivative which contains a C-terminal A fusion protein which is composed of human IL-2 and hirudlin and has both IL-2 activity and hirudin activity. DATED this 25th day of Noveniber 1986. HOEC1HST AKTIENGESE1AISCIAFTF EI)WD) WAliIRS SONS PATENT ATTORNEYS QUEEN STREEIT MELBOURNE. VJIC. 3000. 4 4 4* J S 4~ t~ 4 ~*6S .4 S 4 I I S *5 S 4* 5 0 S @566S5 S *eSe~)4 S U. S U 0 64 0P