IE58935B1 - Fusion proteins with a eukaryotic ballast portion - Google Patents

Abstract

Suitable as "ballast portion" for fusion proteins is a part of the amino-acid sequence of interleukin-2 (IL-2) which comprises considerably less than 100 amino acids. It is advantageous to start from a synthetic IL-2 gene which is divided by unique cleavage sites into six segments of which up to three can be linked in any desired sequence by the building block principle. Specific constructions allowing the solubility of the fusion protein to be varied are possible.

Description

Fusion proteins having a C- or N-terminal portion essentially corresponding to the first 100 amino acids of interleukin-2 have already been proposed (German Patent .

Application P 3,541,856.7), corresponding to European ·» application 86 116140.4 * EP-A2-0,227, 938. The t interleukin-2 portion in these may he derived from mammalian interleukin-2, for example from mouse or rat interleukin-2, which are disclosed in European Patent Application with the publication number (hereinafter EP-A) 0,091»539, but preferably from human interleukia2. These fusion proteins are surprisingly stable in the host cell and can, by reason of their low solubility, easily be separated from the soluble proteins intrinsic to the host.

In a further development of this inventive concept, it has now been found, surprisingly, that even considerably smaller portions of the interleukin-2 molecule are suitable as ballast portion for fusion proteins of this type. The invention consequently relates to fusion proteins having an interleukin-2 (IL-2) portion and a dsired protein, which has the feature that it contains a C— or N-terminal portion which essentially corresponds to the amino acid sequence of IL-2 but which is not biologically active, excepting fusion proteins whose IL-2 portion corresponds essentially at least to the first 100 amino acids of IL-2. Further aspects of the invention are defined in the patent claims. Preferred embodiments are explained in detail hereinafter.

It is particularly advantageous to start from the synthetic gene for human interleukin-2 (hereinafter 15IL-2) which is described in EP-A 0,163,249 and depicted in the addendum. This synthetic gen® contains a number of unique restriction cleavage sites which permit the DNA coding for IL-2 to be broken down into manageable segments.

Using these segments It is possible by the modular principle to tailor the ballast portion for fusion proteins, the solubility of the fusion proteins obtained ranging from high to low depending on the combination of the segments and depending on the nature of the desired protein.

Thus the invention allows the solubility to be directed towards that which is most advantageous for th© possible or desired working up of the product, that is to say high solubility when the product is to be purified by chroma10 tography, for example using an antibody column, or low solubility if, for pre-purification, the soluble proteins intrinsic to the host are to be removed, for example by centrifugation.

A particular advantage of the invention is that It is possible to prepare fusion proteins having a very small ballast portion, since this results in the relative yield of desired protein being considerably increased.

Another advantage of the invention is that the ballast portion can be constructed in such a way that it impairs the spatial structure of the desired protein as little as possible and thus, for example, does not prevent folding up.

Cleavage of the fusion proteins results in not only the desired protein but also the ballast portion", that is to say the IL-2 derivative. This may have il-2 activity (T-cell proliferation test) or bind to IL-2 receptors.

The modular principle according to the invention can thus also be used to produce, as "by-products", IL-2 derivatives which have the biological activities of IL-2 to a greater or lesser extent.

Particularly advantageous embodiments of the invention are explained hereinafter with reference to the synthetic gene described In EP-A 0,163,249. This gene is cut-at the 5 ’ end with the restriction endonuclease EcoRI and at the 3' end with Sail. Apart from the three unique res trie tion cleavage sites for the enzymes Pstl, Xbal and Sad, which were used to construct this gene, the locations of the unique cleavage sites for Hlul and Pvul are also favorable. When the sequences located between these cleavage sites are designated A to ?, the synthetic gene can be represented diagraram&tically as follows (EcoRI)-A-Pstl-S-MluI-C-Xbal-D-SacX-R-PvuI-F-(Sail) Th© segments A to F are thus particularly suitable units for the modular system according to th® invention. Thus, in this representation the ballast portion for the fusion proteins described in German Patent Application P 3,541,856.7 corresponds to the segments A to E, and that for the bifunctional protein having the entire IL-2 gene, which is mentioned in the same application, corresponds to all the segments A to P. In contrast, the gene constructs according to the invention relate to other combinations of the segments A to F, preferably having fewer than 4 of these segments, the segment A coding for the N-terminal end of the fusion protein. The arrangement of th® other segments is arbitrary, use optionally being made of appropriate adaptors or linkers. Appropriate adaptor or linker sequences can also be introduced at the C-terminal end of the ballast portion, and in this case they can code for amino acids or short amino acid sequences which permit or facilitate th® cleavage off, enzymatically or chemically, of the ballast portion from the desired protein. Th® adaptor or linker sequences can, of course, also be used to tailor the ballast portion for a particular fusion protein, for example to achieve a desired solubility. In this contest, it has emerged, surprisingly, that the solubility of the fusion proteins does not depend on th© molecular size but that, on th® contrary, even relatively small molecules may have low solubility. Thus, with knowledge of these relationships, which are explained in detail in the examples, those skilled in the art are able without great experimental effort to obtain fusion proteins according to the invention with a small ballast portion and having particular desired properties.

Thus, if the desired protein is a eukaryotic protein the fusion proteins obtained according to the invention are composed exclusively or virtually exclusively of eukaryotic protein sequences. However, surprisingly, these fusion proteins are not recognised as foreign proteins by the prokaryotic host cells, and are not rapidly degraded by proteases intrinsic to the host. This degradation takes place particularly often in the case of proteins which are foreign to the host and coded tor by cDNA sequences which are to be expressed in bacteria. It has now emerged that cDNA sequences can be expressed very effectively if they are embedded In the segments according to the invention. It is possible to construct specific vectors for this purpose, which contain between the sequences according to the Invention a polylinker sequence which has several cloning sites for the cDNA sequences. Where the cDNA which has been cloned in contains no stop codon the polypeptide sequence coded for by the cDNA sequence Is additionally protected by the polypeptide for which the C-terminal segment codes.

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. If the latter contains, for example, no methionine it Is possible for the connecting element to denote Met, in which case chemical cleavage with cyanogen chloride or bromide is carried out. If there Is a cysteine at the carboxyl terminal end of the connecting element, or if the connecting element represents Cys, then if is possible to carry out a cysteine-specific enzymatic cleavage or chemical cleavage, for example after specific S-cyanylatron. If there Is a tryptophan at the carboxyl terminal end of the bridging element, or if the connecting element represents Trp, then chemical cleavage with N-bromosuccinimide can. be carried out.

Desired proteins which do not contain Asp - Fro in their amino acid sequence and are sufficiently stable to acid can, as fusion proteins with this bridging element, be cleaved proteolytically in a manner known per se. This results in proteins which contain ^-terminal proline or C-terminal aspartic acid. It is therefore also possible in this way to synthesise modified proteins.

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

Examples of enzymatic cleavages are likewise known, it also being possible to use modified enzymes of improved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297). If th© desired eukaryotic peptide is proinsulin, then the chosen sequence is advantageously a peptide sequence in which an amino acid which can be split off with trypsin (Arg, Lys) is bonded to the Siterminal amino acid (Phe) of pro insulin, for example AlaSer-Met-Thr-Arg, since in this ease the arginine-specific cleavage can be carried out with the protease trypsin.

If th® desired protein does not contain the emino acid sequence I1e-Glu-Gly-Arg, then the fusion protein with the appropriate bridging element can be cleaved with factor 2£a (EP-A 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. All known host-vector systems are suitable for this purpose, that is to say, for example, mammalian cells and microorganisms t for example yeasts and, preferably, bacteria, in particular E. coli.

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

In bacterial hosts, it is advantageous to select the promoter and operator from the group comprising lac, tac, trp, PL or PR of phase λ, hsp, omp or a synthetic promoter as proposed in, for example, German Offenlegungsschrift 3,430,683 (EP-A 0,173,149). The tac promoter-operatorsequence is advantageous and is now coamercially available (for example expression vector pKK223-3, Pharmacia, Molecular Biological», Chemicals and Equipment for Molecular Biology·3, 1904, page 63).

In the expression of the fusion protein according to the invention It may prove advantageous to modify individual 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 wall as modifications, deletions or additions of individual amino acids in the IL-2 protein portion, are familiar to those skilled in the art, and the invention likewise relates to them. Elimination of cysteine or replacement of cysteine by other amino acids, in order to prevent formation of undesired disulfide bridges, as is disclosed in, for example, ΞΡ-Α 109,748, may be mentioned by way of example.

Figures 1 to 13 illustrate in the manner of a flow diagram the processes of the syntheses described in'the examples having the same numbers. To facilitate comprehension, the preparation of the starting materials and intermediates has been depicted in Figures A to C. For the sake of clarity the reference numbers in Figures to 13 each start a new decade, thus (11) in Figure 1. Reference numbers of starting materials to which the present application does not relate end with xero, thus, for example, (20) in Figure 2. The figures are not drawn to scale, in particular the scale is expanded appropriately in the region of the polylinker seguences. IL-2 seguences are defined by thick lines, and structural genes for desired proteins are emphasized in other ways.

Figure A gives an overview of the segments A to F according to the invention and of the combination of segments A and B. The starting material is the plasmid pl59/6, whose preparation is described in detail in EP-A 0,163,249 and which is defined by Figure 5 in this publication.

Figure B shows the expression plasmid pEWlOOO, whose preparation is described in German Patent Application P 3,541,856.7 and is shown in Figure 1 therein. This plasmid is opened in the polylinker seguence by appropriate double digestion, this resulting in the linearized plasmids (Exl) to (Bx4).

Figure C shows the preparation of the pUC12 derivative pW226 and of the expression plasmid pW226-l, both of which contain segments A and F separated by* a polylinker :sequence.

Figure 1 shows th® preparation of the pwC12 derivative pKH40 and. of the expression plasmid pK40, which code for fusion proteins in which the protein sequence corresponding to segment A, that is to say the first 22 amino acids of IL-2, is followed by the bridging element Thr-Arg, with subsequently the amino acid seguence of proinsulin.

Figure 2 shows the construction ox the plasmid pSLll and of the expression plasmid pSL12, which code for polypeptides in which the segment A is followed by a bridging element corresponding to polylinker sequences (2) and (20a), with subsequently the amino acid sequence of proinsulin.

Figure 3 shows the construction of the expression plasmid pK50 which codes for a polypeptide in which segments A t and B? that is to say the first 38 emino acids of IL-2, are directly followed by the amino acid sequence of proinsulin.

Figure 4 shows the construction of the expression plasmid pK51 which codes for a polypeptide in which segments A and 3 are followed by a bridging element corresponding to sequences (42) and (41), to which is connected the amino acid sequence of proinsulin.

Figure 5 shows the construction of the expression plasmid pK52 which differs from pK51 by the inserted Mlul linker (51) which codes for the amino acid sequence which permits cleavage with factor Xa. pK52 can also be obtained from pK50 (Figure 3) by cleavage with Mini and introduction of the said Mlul linker.

Figure 6 shows tha construction of the expression plasmid pK53 from pK51 (Figure 4), likewise by introduction of the Mlul linker.

Figure 7 shows the construction of the expression plasmid pSL14 from pSL12 (Figure 2) by introduction of the fragment C into the polylinker. This results in direct attachment of the segment C to the segment A. In the following polylinker th® first two amino acids (each Glu) correspond to amino acids 60 and 61 of IL-2. Thus the IL2 portion is composed of amino acids 1 to 22 and 37 to 61. The subsequent amino acid sequence corresponds to that which is coded for by the plasmid pSL12 (Figure 2).

Figure 8 shows the construction of the expression plasmid pPH31 which codes for a fusion protein in which segments A to C are followed by a bridging element which is represented by sequence (81), with subsequently the amino acid sequence of proinsulin.

Figure 9 shows the construction of the plasmid pK192 which codes for a fusion protein in which segments A and B are followed by methionine and, thereafter, the amino acid sequence of hirudin.

Figure 10 shows the construction of the plasmid PW214 which codes for a fusion protein in which segments A and B are followed by the amino acid sequence which permits cleavage with factor Xa, with subsequently the amino acid sequence of granulocyte/raacrophage colony stimulating factor (CSF).

Figure 11 shows the construction of the expression plasmid pW233 which codes for a fusion protein in which segments A and C (corresponding to amino acids 1 to 22 and 37 to SX of IL-2) are followed, by the bridging element Leu-Thr-He-Asp-Asp-Pro, with subsequently the amino acid sequence of CSF.

Fignre 12 shows the construction of the expression plasmid pW234 which codes for a fusion protein having the following amino acid sequence: Segment A (amino acids 1 to 22) is followed by a bridging element Thr-Arg, then by segment D (amino acids 59 to 96 of IL-2), by Thr-Asp-AspPro as a further connecting element, and finally by CSF.

Figure 13 shows the construction of the plasmids pH20Q and pH201 and of the expression plasmid pH202. These plasmids have a polylinker located between segments A and F or A, B and F, into whose numerous cleavage sites foreign DNA can be cloned. These plasmids are particularly suitable for cloning cDNA sequences.

The invention is explained in detail in the examples which follow, in which th© numbering coincides with that in the figures. Unless otherwise stated, percentage data relate to weight.

Example A The starting plasmid p!59/6 is described in EP-A 0,163,249 (Figure 5). The sequence defined there as WIL2 or in the text as DKA sequence I is in Figure A divided into segments A to F which are bounded by cleavage sites for the enzymes EcoRI, Pstl, Mlul, Xbal, Sad, Pvul and Sail. Double digestion with the appropriate enzymes results in the segments (A) to (?) or adjoined segments, for example the segment (A,B) with EcoRI and Mlul.

Example 3 The preparation of the expression plasmid pEWlOOO has been proposed in the (not prior-published) German Patent Application P 3,541,856.7 (Figure 1). This plasmid is a derivative of the plasmid ptacll (Amann et al». Gene 25 (1983) 167 - 178) into whose recognition site for EcoRI has been incorporated a synthetic sequence which contains a Sail cleavage site. In this way the expression plasmid pKKl/7.3 is obtained. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 - 769) results in the plasmid pJF118. This is opened at the unique restriction cleavage site for Aval, and is, in a known manner, shortened by about 1000 bp by exonuclease treatment and is ligated. This results in the plasmid pEWlOOO. Opening this plasmid in the polylinker using the enzymes EcoRI and Hindlll, Sail, Pstl or Sma I results in the linearized expression plasmids (Exl), (Ex2), (Ex3) and (Ex4).

Example C The eomraere rally available plasmid pUC12 is opened with EcoRl and Sail, and the .linearised plasmid (1) is isolated. Ligation of (1) with the segment (A), the synthetic linker sequence (2) and th© segment (?) results in the plasmid pW226 (3).

The strain E. coli 79/02 is transformed In a known manner with the plasmid 'DNA from the ligation mixture. The cells are plated out on agar plates which contain isopropyl-0D-thiogalactopyranoside (IPTG), 5"bromO"4-chloro-3indolyl-0-D-galactopyranoside (X-gal) and 20 >*g/ml ampicillin (Ap). The plasmid DNA is obtained from white clones, and the formation of th© plasmid (3) is confirmed by restriction analysis and DNA. sequence analysis.

The small EeoRI-Hindlll fragment (4) is cut out of the plasmid (3) and is isolated. This fragment is ligated with the linearised expression plasmid (ExX) in a T4 DNA ligase reaction. The resulting plasmid pW226-l (5) is characterised by restriction analysis.

Competent cells of the strain Ξ. coli Me 1061 axe transformed with DNA from the plasmid pW 226-1. Clones which are resistant to ampicillin are isolated on Apcontaining agar plates. The plasmid DNA Is reisolated from Me 1061 cells and then characterised anew by restriction analysis. Competent cells of the E. coli strain w 3110 are now transformed with plasmid DNA isolated from E. coli Me 1061 cells. E. coli V 3110 cells are always used for expression hereinafter. All the expression experiments in th® stated examples are carried out in accordance with the following conditions.

An overnight culture of E. coli cells which contain the plasmid (5) Is diluted in the ratio of about Is 100 with LB medium (J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) which contains 50 ^g/ml ampicillin, and growth is followed by measurement of the OD. When the OD is 0.5 the culture is adjusted to 1 mM in 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 7.0) for 5 minutes, and samples ar® applied to an SDS gel electrophoresis plate. After electrophoresis, bacteria which contain the plasmid (5) produce a protein band which corresponds to the size of the expected protein (6 kD).

The stated induction conditions apply to shake cultures; for larger fermentations appropriate modifications of the OD values and, where appropriate, slight variations in the IPTG concentrations are advantageous.

The resulting protein shows no biological activity in a cell proliferation test with an IL-2-dependent cell line (CTLL 2)Example 1 The plasmid (3) is opened with Mlul and Sail, and the two resulting fragments are separated by gel electrophoresis. Th® larger fragment (11) is isolated.

The synthetic oligonucleotide (12) is ligated with the blunt-ended DMA (13) coding for proinsulin (Hetekam et al., Gene 19 (1982) 179 - 183), this resulting in DNA sequence (14). The latter is cut with Hlul and Sail, this resulting in DNA sequence (15). The latter is now ligated with the fragment (11), this resulting in formation of the plasmid pKH40 (16). The latter is characterised by restriction analysis.

The plasmid (16) is digested with EcoRI and Hindlll, and the small fragment (17) is isolated by gel electrophoresis. Ligation with the linearised expression plasmid (Sxl) results in the expression plasmid pK40 (18), Expression as indicated in Example C results in a protein which, after cell disruption, is found in the soluble fraction of cellular protein. The Western blot technique is used to demonstrate that the proinsulin sequence is Intact.

Example 2 The starting material is the plasmid pPH30 which is depicted in the (not prior-published) German Patent Application P 3,541,856.7, in Figure 3c. Within the meaning of the present invention, in Figure 2 the IL-2 part sequence is shown as A-E (20). The end of this sequence and the bridging element up to the proinsulin sequence is shown as (20a) in Figure 2.

The plasmid (20) is digested with Pvul and Hindlll, and the small fragment (22) is isolated. In addition, the plasmid (3) is opened with EcoRI and Pvul, and the small fragment (23) is isolated. Moreover, the vector pUC12 is digested with EcoRI and Hindlll, and the large fragment (21) is isolated. Ligation of the fragments (21), (23) and (22) results in the plasmid pSLll (24).

The plasmid (24) is digested with Hindlll and partially with EcoRI, and the fragment (25) which contains the segment A and the proinsulin gene Is isolated. Ligation of (25) into the linearised expression plasmid (Sxl) results in the expression plasmid pSL12 (26).

Expression as indicated in Example C and subsequent working up results in a soluble fusion protein. Western blot analysis with insulin antibodies confirms that this protein contains the intact insulin sequence.

Example 3 Th© plasmid ptrpED5-l (30) (Hallewell et al., Gen® 9 (1980) 27-47) is used for amplification of th© proinsulin gene. The plasmid is opened with Hindlll and Sail, and the large fragment (31) is isolated. The fragment (31) is ligated with DNA sequence (14)» this resulting in the plasmid pH106/4 (32).

Th® plasmid (32) is digested with Sail and Mlul, and the small fragment (15) is isolated. The linearized expression plasmid (Ex2)» the segment (A,B) and the fragment (15) are now ligated, this resulting in the expression plasmid pK50 (33).

Expression of the coded fusion protein is carried out as indicated, in Example C. The cells are then spun down from the culture broth and ruptured In a French press. The protein suspension is now centrifuged to separate it into its soluble and insoluble protein constituents. The two fractions are analyzed by gel electrophoresis in a known manner on 17.5% SDS polyacrylamide gels and subsequently by staining the proteins with th® dyestuff Coomassie blue. It is found, surprisingly, that the fusion protein is located In the insoluble sediment. Western blot analysis with Insulin antibodies confirms that intact proinsulin is present In the fusion protein.

Th® sediment from the French press disruption can now immediately be used further for isolation of proinsulin.

Example 4 The starting material is the plasmid pPH20 (40) which is depicted in German Patent Application P 3,541,856.7» in Figure 3c. Cutting this plasmid with EcoRl» filling in the protruding ends and cutting with Hindlll results In the fragment (41)» from which the DMA sequence of the part of (40) which is of interest here can be seen.

Ligation of the linearised expression plasmid (Sx4) with the segment (A,3), the synthetic oligonucleotide (42) and th® fragment (41) results in the plasmid pK51 (43).

Example 5 Ligation of the linearised expression plasmid (Ex2) with the segment (A,B), the synthetic oligonucleotide (51) and the DNA sequence (15) results in the plasmid pK52 (52). The correct orientation of the oligonucleotide (51) is established by sequence analysis. The plasmid codes for a fusion protein which contains the amino acid sequence which corresponds to the oligonucleotide (51) and thus can be cleaved by activated Factor Za.

The plasmid (52) can also be obtained in the following manner; Partial cutting of the plasmid (33) with Mlul and ligation of the resulting opened plasmid (53) with the DNA sequence (51) likewise results in the plasmid pK52.

Example 6 Partial cutting of the plasmid (43) with Mlul and. ligation of the resulting linearised plasmid (61) with the synthetic DMA sequence (51) results in the plasmid pK53 (62). The latter likewise codes for a fusion protein which can be cleaved with activated factor la. The correct orientation of the sequence (51) is established, as in Example 5, by DNA sequence analysis.

Example 7 The plasmid (26) is cleaved with Xbal and partially with Mlul, and the large fragment (71) is isolated. Ligation with the segment (C) results in the plasmid pSL14 (72).

After expression and cell disruption, the fusion protein is found in the soluble fraction of cellular protein.

Example 8 The plasmid (20) is cleaved with Sbal and partially with EcoRI, and the protruding ends are filled in, this resulting in DNA sequence (81). Ligation under blunt end conditions results in the plasmid pPH31 (82). The fusion protein is found in the insoluble fraction of cellular protein.

Example 9 The starting material used is the plasmid (90) which is described in EP-A 0,171,024 (Figure 3). This plasmid is reacted with Sail and then with AccI, and the small fragment (91) is isolated. The latter is ligated with the synthetic oligonucleotide (92), this resulting in DNA sequence (93). The latter is cut with Mlul, this resulting in DNA fragment (94).

The plasmid (33) is digested with Mlul, partially, and with Sail, and the large fragment (95) is Isolated. Th® latter is ligated with the DNA. sequence (94), this resulting in the expression plasmid pKl92 (96). The latter codes for a fusion protein in which the first 38 amino acids of IL-2 are followed by methionine and then by the amino acid sequence of hirudin. The fusion protein is found in the soluble fraction of cellular protein.

Example 10 The starting material used is the plasmid pHG23 (100) which Is described in EP-A. 0,183,350 and which Is generally accessible from the American Type Culture Collection under No. ATCC 39000. This plasmid is cut with SfaNI, the protruding ends ar® filled in, then reaction with Pstl is carried out, and the small fragment (101) is isolated. Ligation of the linearised expression plasmid (2x3) with the segment (A,B), the synthetic oligonucleotide (102) and the fragment (101) results in the expression plasmid pW214(103). This plasmid codes for a fusion protein in which the first 38 amino acids of IL-2 are followed by the sequence which is derived from the oligonucleotide (102) and which allows the molecule to be cleaved with factor Xa, with subsequently the amino acid sequence of CSF. After cell disruption, th® fusion protein is found in the insoluble fraction of cellular protein.

Example 11 The starting plasmid pw21S (110) is proposed in German Patent Application P 3,545,568.3 (Figure 2b). In this plasmid, the IL-2 sequence corresponding to segments A to E (Pvul cleavage site) is followed by a linker which codes for the amino acids Asp-Asp-Pro, immediately followed by the amino acid sequence for CSF. The connecting sequence between IL-2 and CSF allows the fusion protein to be cleaved proteolytically.

The sequence (111) is isolated from the plasmid (110) by cutting with Pvul and Hindlll.

The plasmid (3) is cut with Mlul and Xbal, and the large fragment (112) is isolated. The latter is ligated with the segment (C), this resulting in the plasmid pW227 (113). This plasmid is reacted with EcoRI and Hindlll, and the short fragment (114) is Isolated. If this fragment is ligated with the linearised, expression plasmid (Exl) the result is the plasmid pw227~l (115). The plasmid codes for a protein which is derived from IL2 but which has no IL-2 activity.

The plasmid (113) is additionally cut with ScoRI and Pvul, and the short fragment (116) Is isolated. Ligation of the linearised expression plasmid (Exl) with the fragments (116) and (111) results In the expression plasmid pW233 (117). The latter codes for an insoluble fusion protein which, by reason of the abovementioned linker, can be cleaved proteolytically.

Example 12 The plasmid (3) is cut with Xbal and Sacl, and. the large fragment (121) is isolated. Ligation with the segment (D) results in the plasmid pW228 (122). Th® latter is cut with EcoRl and Hindlll, and the small fragment (123) is isolated. Ligation of the linearized expression plasmid (Exl) with the fragment (123) results in the expression plasmid pW228-l (124). This plasmid codes for a biologically inactive IL-2 derivative. The plasmid is digested with EcoRl and Pvul, and th® short fragment (125) is isolated. Ligation of the linearised expression plasmid (Exl) with the fragments (125) and (111) results in the expression plasmid pW234 (126). The latter codes for a sparingly soluble fusion protein which can likewise be cleaved proteolytically.

Example 13 For the construction of plasmids which are suitable, in particular, for the expression of cDNA sequences, initially the polylinker sequence (131) is synthesised.

Ligation of the linearized plasmid (1) with the segment (A), the polylinker sequence (131) and segment (?) results in the plasmid pH200 (132).

The plasmid (132) is reacted with EcoRl and Mlul, and the large fragment (133) is Isolated. Ligation of the latter with the segment (A,B) results in the plasmid pH201 (134).

The plasmid (134) is reacted with EcoRI and Hindlll, and the short fragment (135) is isolated. Ligation of this fragment with the linearised expression plasmid (Exl) results in the expression plasmid pK 202 (13S).

The plasmid (135) is opened with BamHI, and the cDNA which is to be expressed is introduced into the linearised plasmid via a commercially available BamHI adaptor. Depending on the orientation of the cDNA, every third sequence is attached to (A,B) in the reading frame.

If the cDNA sequence contains no stop codon the polypeptide sequence for which it codes is additionally protected by the amino acid sequence corresponding to the segment (F).

If the cDNA is not connected in the correct reading frame, a shift of the reading tram® is brought about by, for example, cleaving the cDNA-containing (original or multiplied) plasmids with Mlul or Xbal (as long as the cDNA does not contain cleavage sites for these enzymes) and filling in the protruding ends by a Klenow polymerase reaction.

Addendum; DMA Sequence I Triplet Mo. 0 1 2 Amino acid Het Ala Pro Nucleotide No. 1 10 Cod. strand 5' TTC ATG GCG CCG Non-cod. strand 3* G TAG CGC GGC (EcoRI) 3 4 5 6 7 8 9 10 11 12 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 50 60 70 GAA CTG GAA CAC CTG CTG CTG GAC CTG CAG GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC Pstl 23 24 25 26 27 28 29 30 31 32 Met Xie Leu Asn Gly IX© Asn Asn Tyr Lys 80 90 100 ATG ATC CTG AAC GGT ATC AAC AAC TAG AAA TAG TAG GAC TTG CCA 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 110 120 130 AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC TTG GGC TTT GAC TGC GCA TAG GAC TGG AAG Mini „01 _ I»» 4 3 Λ 45 46 Lys Phe 140 Tyr Met Pro §4 Ia 1ft ΛΛΛ Φ|^ ζ-a ATG CCG ATG TAG GGC 53 54 55 56 57 Leu i^ys 170 His Leu Gln CTG AAA Ρ&Γ· CTC GAC fprprp GTG GAG GTC 48 49 50 51 52 Lys Lys Ala Thx Glu 150 160 7ft 7ft % 1ft IR GCT ACC tsj-asgyej^ rp - CGA TGG ριίγ&ίρ «Κ® X Μ 58 ςα eJ afi 60 61 62 Cys Leu Glu OX is Glu 180 190 TG'·’ γφ* W* ΑΛΑ f*> m *r GAG ACA z*> 1¾ m £*epfp CTC Xbax S3 £ U ‘'S >" £-» o5 e> s* SO 67 68 69 70 71 72 jjen Lys Pro Leu Glu Glu Val Leu Asn bSU 200 210 220 CTG % % CCG CTG GAG GAA CTG CTG fJSfs tp^rp GGC GAC c^c ^φφ L> A A GAC GAC 73 74 75 76 77 78 79 80 81 82 Ai ci Gln Ser Lys Asn Phe His Leu &T*er Pro 230 240 250 W*» a φΓ*φ AttfA £ τ» 1» * φ ίΠ4«Α W ·» W CGT CCG CGA GTC 1ft *?» Gp?f3?p φφΤΚ GTG GAC nna GGC 83 84 85 86 87 88 89 90 91 92 Arg Asp Leu II© Ser Asn lie & (S> 3*1 Val He 260 270 280 CGT ns. π ««rAibt» CTG· ATC AAC ϊ\ Φ0 1ft 1ft /*J GTT ^PC9 q&C Sh 1¾ TTG TAG /-*> «1 IK TAG 93 94 95 96 97 98 0 Q a>> *r 100 ini V A 102 Val Leu Glu Leu Lys Gly Ser Glu *»* A nSaOa Thr 290 300 310 GTT CTG GAG CTC AAA GGT ΊΆ ACC ACG Λ ?l ?1 GAC CTC ρρφφ CCA 7ft f**X ptfpp TGG TGC Sac I 103 104 105 105 107 108 109 110 1 1 Ί 112 ΡΗθ Met 320 Cys Glu Tyr Ala 330 Asp Glu Thr *3 rt Ala ejse^iQ %ι m/·8 Aid? TGC ££ ΐιc GCG /•tip pnw A GCG AAG TAG ACG CCT ATG CGC CTG CGC 113 114 115 116 117 118 119 120 121 122 Thr II© Val Glu Phe Leu Asn Arg TXTsO II© 350 360 370 ACG pfpftl Ο?Ί «Α» PpPprp AAC CGT TGG· ATC TGC CAA ^bpfp A. 0>%W qwpg GCA ACC TAG Pvul 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 ACC CTG TGG AAG ACG GTC AGC TAG TAG AGA TOG P*P Isr&L» 133 134 135 410 ACC TAG 3» TGG ACT ATC AGC 5e (Sail)

Claims (21)

Patent Claims

1. A fusion protein having an interleukin-2 (IL-2) portion and a desired protein, which has the 5 feature that it contains a C- or N-terminal portion .which essentially corresponds to the amino acid sequence of IL-2 but which is not biologically active, excepting fusion proteins whose IL-2 portion corresponds essentially at least to the 10 first 100 amino acids of IL-2.

2. A fusion protein as claimed in claim 1, wherein the amino acid sequence corresponds to' that of human IL-2.

3. A fusion protein as claimed in claim 2, wherein the 15 gene coding for IL-2 contains part of DNA sequence I (addendum).

4. A fusion protein as claimed in claim 3, wherein the gen® coding for the IL-2 portion is essentially composed of one, two or three of the segments A to 20 P of the IL-2 gene (EcoRl) ~A-PstI-3-KluI-C-XbaI-D-SacI-E-PvuX-F~ (Sail) in arbitrary sequence, where appropriate linked via adaptor or linker sequences.

5. A fusion protein as claimed in one or mor© of the 25 preceding claims, wherein is located, between the IL-2 sequence and the amino acid sequence of the desired protein, an amino acid or amino acid sequence which allows the desired protein to be cleaved off, chemically or enzymatically, from the 30 IL-2 portion. δ. A fusion protein as claimed in claim 5, wherein the amino acid is Met, Cys, Trp, Lys or Arg, or the amino aeid sequence contains these amino acids at the C-terminal end.

6. 7. A fusion protein as claimed in claim 6, wherein the amino acid sequence is Asp-Pro or contains this amino acid sequence at the C-terminal end.

7. 8. A fusion protein as claimed in claim 6, wherein the amino acid, sequence is Ile-Glu-Gly-Arg or contains this amino acid sequence at the C-terminal end.

8. 9. A process for the preparation of the fusion proteins as claimed in one or more of claims 1 to 8, which comprises causing the expression of a gene coding for the fusion protein in a host cell.

9. 10. The process as claimed in claim 9, wherein the gene is incorporated in an expression vector and is expressed in a bacterial cell.

10. 11. The process as claimed in claim 10, wherein the bacterial cell used is E. coli.

11. 12. The use of the fusion proteins as claimed in claims 1 to 8, or of the fusion proteins obtainable as claimed in claims 9 to 11, for the preparation of the desired proteins.

12. 13. A gene structure coding for a fusion protein as claimed in claims 1 to 8.

13. 14. A vector containing a gene structure as claimed in claim 13.

14. 15. The pUC12 derivatives pW226 (Fig. C), pW227 (Fig. 11), pW228 (Fig. 12), pH 200 and pH 201 (Fig. 13), expression plasmids pW226~X (Fig. C), pW227-l (Fig. 11), pW228~I (Fig. 12) and pH 202 (Fig. 13)j expression plasmids coding fox a fusion protein composed of an 11,-2 portion as claimed in claim 1, of a bridging element and proinsulin; pK40 (Fig. 1), pSL12 (Fig. 2), pK50 (Fig. 3), pK51 (Fig. 4), pK52 (Fig. 5), pK53 (Fig. 6), pSL14 (Fig. 7), pPH31 (Fig. 8)? expression plasmid coding for a fusion protein composed, of an IL-2 portion as claimed in claim 1, of a bridging element and hirudin: pK192 (Fig. 9)- expression plasmids coding for a fusion protein composed of an IL-2 portion as claimed in claim 1, of a bridging element and GM-CSF: pW214 (Fig. 10), pW233 (Fig. 11), pW234 (Fig. 12).

15. 16. A host cell containing a vector as claimed in claim 14 or 15.

16. 17. A fusion protein according to claim 1, substantially as hereinbefore described and exempli tied.

17. 18. A process for the preparation of a fusion protein according to claim 1, substantially as hereinbefore described and exemplified.

18. 19. A fusion protein according to claim 1, whenever prepared by a process claimed in a preceding claim.

19. 20. Use according to claim 12 of a fusion protein, substantially as hereinbefore described and exempli f ied. -2621. A gene structure according to claim 13, substantially as hereinbefore described and exemplified.

20. 22. A vector according to claim 14, substantially as hereinbefore described and exemplified.

21. 23. A host cell according to claim 16, substantially as hereinbefore described and exemplified.