IE863334L

IE863334L - Fusion proteins with a eukaryotic ballast portion

Info

Publication number: IE863334L
Application number: IE863334A
Authority: IE
Original assignee: Hoechst Ag
Priority date: 1985-12-21
Filing date: 1986-12-19
Publication date: 1987-06-21
Also published as: NO865192D0; FI865187L; AU6676086A; JP2774260B2; NO175003C; ATE78296T1; EP0229998A2; DK168823B1; NO865192L; JPS62167799A; KR870006187A; EP0229998A3; IL81018A0; CA1339894C; KR950000301B1; DE3636903A1; IE58935B1; IL81018A; JP2553058B2; DK619186D0

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

58935 Fusion proteins having a C- or N-termin&l portion essentially corresponding to the first 100 amino acids of interleukin-2 have already been proposed (German Patent .

Application P 3,541,356.7), corresponding to European '» S application 86 116140.4 « EP-A2-0,227,93S. The ? interleukin-2 portion in these may be derived from mammalian interleukin-2, for example from mouse or rat interleukin-2, which are disclosed im, European Patent Application with the publication number (hereinafter 10 "EP-A") 0,091,539, but preferably from human interleukin- 2. 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 20 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 25 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 froai the synthetic gene for human interleukin-2 (hereinafter "'IL-2") which is described in EP-A 0,163,249 and depicted in the addendum. This synthetic gen® contains a number of unique 35 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 indention allows the solubility to be directed towards that which is most advantageous for the possible or desired working up of the product, that is to say high solubility when the product is to be purified by chromatography, 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" s. 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 restriction cleavage sites for the enzymes PstI, 2EbaI and SacI, which were used to construct this gene, the locations o£ the unique cleavage sites for Hlul and Pvul are also 5 favorable. When the sequences located between these cleavage sites are designated A to F, the synthetic gene can be represented diagrammatically as follows (EcoRI) -A-PstI-3-MluI-C~XbaI-D-SacI-S-PvxiI-?- {Sail) The segments A to F are thus particularly suitable "units" for the modular system according to the 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 15 segments A to E, and that for the bifunctional protein having the entire IL-2 gene,, which is mentioned in the sajme application, corresponds to all the segments A to F. In contrast, the gene constructs according to the invention relate to other combinations of the segments A 20 to Fj preferably having fewer than 4 of these segments, the segment A coding for the N-terminal end of the fusion protein- The arrangement of the other segments is arbitrary, use optionally being made of appropriate adaptors or linkers. Appropriate adaptor or linker 25 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 parsait or facilitate the cleavage off, enzymatically or chemically, of the "ballast portion" from the desired protein. The 30 adaptor or linker sequences can, of course, also be used to tailor the "ballast portion" for a particular fnsion 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 the 35 molecular size but that, on the 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 for by cDNA sequences which are to be expressed in bacteria. It has now emerged that cDMA, 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 cDMA which has been cloned in contains no stop codon the polypeptide sequence coded for by the cDMA sequence is additionally protected by the polypeptide for which the C--terminal segment codes.

The fusion protein can be cleaved chemically or ensym&ti-cally 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 outs If there is a cysteine at the c&rboxyl terminal end of the connecting element, or if the connecting element represents Cys, then it is possible to carry out a cysteine-specific enzymatic cleavage or chemical cleavage, for example after specific S-cyanylation. 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 - Pro in their amino acid sequence and are sufficiently stable to acid can, as fusion proteins with this bridging element, be cleaved proteolytics!ly in a manner known per s@. This results in proteins which contain N-terminal proline or 10 C-terminal aapartic acid. It is therefore also possible in this way to synthesize 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-Pro, n 15 denoting 1 to 3.

Examples of enzymatic cleavages are likewise knows.£, it also being possible to use modified enzymes of improved specificity (cf. C.S. Craik et al-, Science 228 (1985) 291-297), If the desired eukaryotic peptide is proinsulin, then the chosen sequence is advantageously a peptide sequence in which an amino acid which can b© split off with trypsin (Arg„ Lys) is bonded to the M~ terminal amino acid (Phe) of proinsulin, for example Ala-25 Ser-Met-Thr-Arg, since in this case the argin£ne~specific cleavage can. be carried omt with the protease trypsin.

If the desired protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, then the fusion protein with the appropriate bridging element can be cleaved with factor la (EP-A 0,025,190 and 0,161,973)- "S^he 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, for example yeasts and t, preferably, bacteria,, in particular E. coli.

The DMA seqtien.ce which codes for the desired protein is 5 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 10 promoter and operator from the group comprising lac, tac, trp, or PR of phase A, hsp, omp or a synthetic promoter as proposed in, for example, German Offenlegungsschrift 3,.430,683 (EP-A 0,173,149). The tac promoter-oper&tor-seguence is advantageous and is now commercially 15 available (for example expression vector pKX223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63).

In the expression of the fusion protein according to the 20 invention it may prove advantageous to modify individual triplets for the first few amino acids downstream of the A.TG start codon in order to prevent any base-pairing at the ftiBMA level. Modifications of this type,, as well as modifications j, deletions or additions of individual amino 25 acids in the IL-2 protein portion, are fasalllar to those skilled in the art, and the invention likewise relates to them. Elimination of cysteine or replacement of cysteine by other amino acidsf in order to prevent formation of undesired disulfide bridges, as is disclosed in, for 30 example, 3P-A 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 35 examples having the same numbers. To facilitate comprehension? the preparation of the starting materials and intermediates has been depicted in Figures h to C. For the sake of clarity the reference numbers in Figures _ 7 - 1 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 zero, thus, for example ,, (20) in Figure 2. The figures are not drawn 5 to seal®,, in particular the scale is expanded appropriately in the region of the polylinker sequences. IL-2 sequences 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 15 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 20 plasmid is opened In the polyl inker sequence by appropriate double digestion, this resulting in the linearized plasmids (Exl) to (Ex4)- Figure C shows the preparation of the pUC12 deriiratitre 25 pW226 and of the expression plasmid pW226-l, both of which contain segments A and F separated by a polvlinker sequence.

Figure 1 shows the preparation of the pUC12 derivative 30 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 sequence of 35 proinsulin- Figure 2 shows the construction of the plasmid pS&ll 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 3, that is to say the first 38 amino acids of IL-2, are directly followed by the amino acid sequence of 10 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 15 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 pKSl by the inserted Mlul linker 20 (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 Mlul and introduction of the said Mlul linker.

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

Figure 7 shows the construction of the expression plasmid 30 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 polyl inker the first two amino acids (each Glu.) correspond to amino acids 60 and 61 of IL-2- Thus the IL-35 2 portion is composed of amino acids 1 to 22 and 3/ 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 5 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 10 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 15 cleavage with factor Xa, with subsequently the amino acid sequence of granulocyte/macrophage colony stimulating factor (CSF).

Figure 11 shows the construction of the expression 20 plasmid pW233 which codes for a fusion protein in which segments A and C (corresponding to amino acids 1 to 22 and 37 to 61 of IL-2) are followed by the bridging element Leu-Thr-Ile-Asp-Asp-Pro, with subsequently the amino acid sequence of CSF.

Figure 12 shows the construction of the expression pl&smid pW234 which codes for a fusion protein having the following amino acid sequences Segment A (amino acids 1 to 22) is followed by a bridging element Thr-Arg, then by 30 segment D (amino acids 59 to 96 of IL-2), by Thr-Asp-Asp-Pro 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 35 plasmids have a polylinker located between segments A and F or A, B and ¥, 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 the numbering coincides with that in the figures. Unless otherwise stated, percentage data relate to weight.

Example A The starting plasmid pl59/6 is described in EP-A 0,163,249 (Figure 5). The sequence defined there as WIL-10 2" or in the text as "DMA sequence I" is in Figure A divided into segments A to F which are bounded by cleavage sites for the enzymes EcoRI, Pstl, Mlul, Sbal, Sac I, Pvul and Sail. Double digestion with the appropriate enzymes results in the segments (A) to (F) or 15 adjoined segments, for example the segment (A,S) with EcoRI and Mlul.

Example B 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 25 has been incorporated a synthetic sequence which contains a Sail cleavage site. In this way the expression plasiaid pKX177.3 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 30 cleavage site for Aval, and is, in a known manner, shortened by about 1000 bp by exonucle&se treatment and is ligated. This results in the plasmid pEMlOOO. Opening this plasmid in the dolylinker using the enzymes EcoRI and Hindi!I, Sail, PstI or Smal results in the linearised 35 expression plasmids (Exl), (Ex2), (Ex3) and (Ex4).

Example C The commercially available plasmid pUC12 is opened with EcoRI and Sail/ and the .linearised plasmid (1) is 5 isolated. Ligation of (1) with fche segment. (A), the synthetic linker sequence (2) and -the segment (F) results in the plasmid pW226 (3).

The strain. E. coli 79/02 is transformed in a known manner 10 with the plasmid DMA from the ligation mixture. Til© cells are plated out on agar plates which contain isopropyl-0-D-thiogalactopyranosid® (IPTG), 5-bromo-4-chloro-3-indolyl-/9~D~galactopyranoside (X-gal) and 20 pg/ml ampicillin (Ap). The plasmid DNA is obtained from white 15 clones, and the formation of the plasmid (3) is confirmed by restriction analysis and DNA sequence analysis.

The small EcoRI-Hindi 11 fragment (4) is cut out of the plasmid (3) and is isolated. This fragment is ligated 20 with the linearized expression plasmid (Exl) in a T4 DMA ligase reaction. The resulting plasmid pW226-l (5) is characterized by restriction analysis.

Competent cells of the strain E. coli He 1061 are 25 transformed with DMA from the plasmid pW 226-1. Clones which are resistant to ampicillin are isolated on. Ap-containing agar plates. The plasmid DNA is reieolated from Mc 1061 cells and then characterised anew by restriction analysis. Competent ceils of the E. coli 30 strain W 3110 are now transformed with plasmid DNA isolated from E. coli Mc 1061 cells. E. coli W 3110 cells are always used for expression hereinafter. All the expression experiments in the stated examples ar© 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 100 minutes, 5 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 are applied to an SDS gel electrophoresis plate. After electrophoresis,, bacteria which contain the plasmid (5) 10 produce a protein band which corresponds to the sise of the expected protein (6 kD).

The stated induction conditions apply to shake cultures; for larger fermentations appropriate modifications of the 15 OD values and, where appropriate, slight variations in the IPTG concentrations are advantageous.

The resulting protein sho%?s 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 25 resulting fragments are separated by gel electrophoresis.

The larger fragment (11) is isolated.

The synthetic oligonucleotide (12) is ligated with the blunt-ended DNA (13) coding for proinsulin (Wetekam et 30 &1., Gene 19 (1982) 1/9 - 183), this resulting in DNA sequence (14). The latter is cut with Mlul and Sail, this resulting in DMA sequence (15). The latter is now ligated with the fragment (11),, this resulting in formation of the plasmid pKH40 (16)- The latter is characterized by 35 restriction analysis.

The plasmid (16) is digested with EcoEI and HindiII, and the small fragment (17) is isolated by gel electrophoresis. Ligation with the linearised expression plasmid (Exl) 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 5 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 15 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 HindiII, and 20 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 HindiII, and the large fragment (21) is isolated. Ligation of the fragments (21), (23) 25 and (22) results in the plasmid pSLll (24).

The plasmid (24) is digested with Hindi 11 and partially with EcoEI, and the fragment (25) which contains the segment Jk and the proinsulin gene is isolated. Ligation 30 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 35 blot analysis with insulin antibodies confirms that this protein contains the intact insulin sequence. 1 Ji Example 3 The plasmid ptrp3D5~l (30) (Kallewell et al.„ Gen® 9 (1980) 27-47) is used for amplification of the proinsulin 5 gene. The plasmid is opened with HindiXI and Sail, and the large fragment (31) is isolated. The fragment (31) is ligated with DMA sequence (14), this resulting in the plasmid pHlQS/l (32)* The 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 20 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 the dyestuff CooaiassI® blue. It is found, surprisingly, that the fusion protein 25 is located in the insoluble sediment. Westersi blot analysis with insulin antibodies confirms that intact proinsulin is present in the fusion protein.

The sediment from the French press disruption can now 30 immediately be used further for isolation of proinsulin.

Example 4 The starting material is the plasmid pPH20 (40) which is 35 depicted in German Patent Application P 3,541,856.7, in Figure 3c. Cutting this plasmid with EcoRI, filling in the protruding ends and cutting with HindiII results in the fragment (41), from which the DNA sequence of the part of (40) which is of interest here can be seen.

Ligation of the linearised expression plasmid (Ex4) with the segment (A,B), the synthetic oligonucleotide (42) and 5 the fragment (41) results in the plasmid pK51 (43).

Example 5 Ligation, of th© linearised expression plasmid (Ex2) with 10 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 (31) is established by sequence analysis. The plasmid codes for a fusion protein which contains the amino acid sequence 15 which corresponds to the oligonucleotide (51) and thus can be cleaved by activated Factor la.

The plasmid (52) can also be obtained in the following manners Partial cutting of the plasmid (33) with Mlul and ligation of the resulting opened plasmid (53) with the DMA sequence (51) likewise results in the plasmid pX52.

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 (52). The latter likewise codes for a fusion protein 30 which can be cleaved with activated factor Xa. 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 Mlult 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 Xbal 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 10 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 DMA 20 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. The 25 latter is ligated with the DNA sequence (94), this resulting in the expression plasmid pK192 (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 30 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 Sf&NI, the protruding ends are filled in, then reaction with PstI is carried out, and the small fragment (101) is isolated. Ligation of the linearised expression plasmid (Ex3) with the segment (A,3)„ the synthetic oligonucleotide (102) and the fragiaent (101) results in the expression plasmid 5 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 10 CSF. After cell disruption,, the fusion protein is found in the insoluble fraction of cellular protein.

Example 11 The starting plasmid pW216 (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 sit®) is followed by a linker which codes for the amino acids Asp-Asp-Pro, immediately 20 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 25 cutting with Pvul and HindiII.

The plasmid (3) is cut with Mlul and Xb&Z, and the large fragment (112) is isolated. The latter is lig&ted with the segment (C)this resulting in the plasmid pW227 30 (113). This plasmid is reacted with EcoRI and HindXII, and the short fragment (114) is isolated. If this fragment is ligated with the linearized expression plasmid (Exl) the result is the plasmid pW227~l (115)., The plasmid codes for a protein which is derived from IL-35 2 but which has no IL-2 activity.

The plasmid (113) is additionally cut with EcoRI 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 Sac If and. the large 10 fragment (121) is isolated. Ligation with the segment (D) results in the plasmid pW228 (122). The latter is cut with EcoRI and HindiII, and the small fragment (123) is isolated. Ligation of the linearized expression plasmid (Exl) with the fragment (123) results in the expression 15 plasmid pW228-l (124). This plasmid codes for a biologically inactive IL-2 derivative. The plasmid is digested with EcoRI and Pvul, and the short fragment (125) is isolated. Ligation of the linearised expression plasmid (Exl) with the fragments (125) and (111) results 20 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 cDN& sequences,, initially the polylinker sequence (131) is synthesized.

Ligation of the linearized plasmid (1) with the segsaent.

{A), the polylinker sequence (131) and segment (F) results in the plasmid pH200 (132).

The plasmid (132) is reacted with EcoRI and Mlul, and the 35 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 Hindi 11, and the short fragment (135) is isolated. Ligation of this fragment with the linearised expression plasmid (Exl) results in the expression plasmid pH 202 (136).

The plasmid (13S) is opened with BamBI „ 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 cDN&, every 10 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 frame is brought about byt, for example, cleaving the cDNA-containing (original or multiplied) plasmids with Mlul or Xbal (as long as the 20 cDNA does not contain cleavage sites for these enzymes) and filling in the protruding ends by a XIenow polymerase reaction. - 20 Addendum; DMA Sequence I Triplet Mo. Amino acid Nucleotide Mo. Cod. strand Non-cod. strand 3 Thr ACC TGG 13 Gin CAA GTT 4 Ser 20 TC.T AGA 14 Leu 50 CTG GAC Ser TCT AGA ' 3f aa TTC G 0 Met ATG TAC (EcoRI) 6 Ser TCT AGA 7 Thr ACC TGG Lys 30 AAA TTT 9 Lys AAG TTC Thr ACT TGA. 1 Ala GCG C6C 11 Gin 40 CAA GTT 2 Pro CCG GGC 12 Leu CTG gac 16 17 18 19 20 21 22 Glu His Leu Leu Leu Asp Leu Gin 60 70 GAA CAC CTG CTG CTG GAC CTG CAG CTT GTG GAC GAC GAC CTG GAC GTC 23 24 25 26 27 28 2§ 30 Met lie Leu Asn Glv II® Asn Asn 80 90 ATG ATC CTG AAC GGT ATC AAC AAC TAC TAG GAC TTG CCA TAG TTG TTG Pstl 31 32 Tyr Lys 100 TAC AAA ATG TTT 33 A3n AAC TTG 34 Pro 110 CCG GGC Lys AAA TTT Lea CTG GAC 37 38 Thr Arg 120 ACG TGC CGT GCA 39 Met ATG TAC 40 Leu CTG GAC JB *3 X Thr 130 ACC TGG 42 Phe TTC AAG Mlul 4 3 <& wl 45 46 & 7 48 49 50 51 52 Lys Phe Tyr Met.

Pro Lys Lys Ala Glu 140 150 160 * 1A jsuwurfc &TG CCG w t«i «. '*i 3k ACC /"® Ufc ■& pp?p?p H 2aQ SfpQ TAC GGC grgg^^i tf|W|wp CGA TGG 53 54 55 56 57 58 J «« 60 61 62 Lew LsWS His Leu Gin Cys Leu Glu Glu Glu 170 180 190 CTG AAA £»C •«<#•»• «A%*» CTC LPM* fp^qi CTA GAA fa In «& IstfM £-*•&\ f* GAC rprfirp GTG GAG GTC ga pisffl ^fpsp pepfp CTC Xb&i 63 c A Q <*± 65 r> f* 00 67 68 69 70 71 72 ij@U Lys Pro Leu Glu Glu Val Leu S,c;wi «•>%«-# A A Lsu 200 210 220 CTG % ^ ^ CCG CTG GAG CTG CTG fgrgrp GGC GAC CTC OP gi GAC eptfiig GAC 73 74 75 76 77 78 79 80 81 82 Al Gin Ser Lys «r>s>e9 A A Pb® His Leu r<£BS» *WJ Pro 230 240 250 /v'fp UVf A 0^ G ipg* %■ & Bl ^pffjWT j[ CAC CTG CGT CCG CGA GTC MstA sfwp*i 9i GTG GAC era GGC 83 84 85 86 07 88 09 90 91 92 Arg Asp Leu lie Ser As ix lie !& OA.

Val 11© 260 270 280 CGT f2£.f* W*aW CTG ATC *ft «i /*?> a'ff'dj GTT GCA ^ff90 Q&JC Kf3 QJ *\ /"SI ^ §^■£"£^31 J. Afar 93 94 95 96 97 98 CI o f*» ^ 100 i rn V «&> 102 Val Leu Glu lieu Lys Gly S&3T Glu <»•» Thr 290 300 310 GTT CTG GAG CTC AAA GGT f**Si *i ACC ACG "h UJT&A GAC CTC GAG .VftipiTp rrs ft #""3 94 TGG TGC Sac I ~ 22 - 103 104 105 106 107 108 109 110 ill 112 Phe Met Cys Glu Tyr Ala Asp Glu Thr Al& 320 330 340 {pepQ ATG TGC GAA TAC GCG tat f ft ft 4&L> J.

GCG a * A%£%\3! TAC ACG CCT ATG CGC CTG TGA CGC 113 114 115 116 117 118 119 120 121 122 Thr 11® Val Glu Phs Leu Asa Arg Trp lie 350 360 370 ACG Qfpfjl s, tfpifpFp CTG j?xrJL» CGT TGG ATC TGC TAG CAA CTT ^ S.

C3i^ C» bTJI|,PQ GCA ACC TAG Pvul 123 124 125 126 127 128 129 130 131 i 32 AMI A Thr Phe Cys Gin Ser lie lie Ser Thr Leu 380 390 400 ACC TGC CAG TCG ATC ATC ACC CTG TGG AAG GTC AGC TAG TAG AGA TGG GAC 133 134 135 fpVi f 410 srr nnr:a 6> A *k«i. •« A TGG ACT ATC AGC (Sail)

Claims

- 23 - 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 gene coding for the IL-2 portion Is essentially composed of one, two or three of the segments A to 20 F of the IL-2 gene (EcoRI) -A-Pstl-B-Mlul-C-Xbal-D-Sacl-E-Pvul-F- (Sail) in arbitrary sequence, where appropriate linked via adaptor or linker sequences.

5. A fusion protein as claimed in one or more 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. - 24 ~

6. A fusion protein as claimed in claim 5, wherein the amino acid is Met, Cys,, Trp, Lys or Arg, or the amino acid sequence contains these amino acids at the C-terminal end.

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.

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.

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 e gene coding for the fusion protein in a host cell™

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

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

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.

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

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

15. The pUC12 derivatives pW226 (Fig. C), pW227 (Fig. 11), pW228 (Fig. 12), pH 200 and pH 201 - 25 - (Fig. 13) ? expression plasmids pW22S~l (Fig. C), pW227-1 (Fig. 11), pW228~l (Fig. 12) and pH 202 (Fig. 13); expression plasmids coding for a fusion protein composed of an IL-2 portion as claimed in 5 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 10 claimed in claim 1, of a bridging element and hirudin: pK192 (Fig. 9)z 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) f pW234 15 (Fig. 12).

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

17. A fusion protein according to claim 1» substantially as hereinbefore described and 20 exemplified.

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

19. 10. A fusion protein according to claim 1, whenever prepared 25 by a process claimed in a preceding claim.

20. Use according to claim 12 of a fusion protein, substantially as hereinbefore described and exemplif ied. -26-

21. A gene structure according to claim 13, substantially as hereinbefore described and exemplified.

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

23. A host cell according to claim 16, substantially as hereinbefore described and exemplified- F. R. KELLY & CO. , AGENTS FOR THE APPLICANTS.