FI93734B - Process for producing fusion proteins containing a eukaryotic ballast fraction and products useful in the process - 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

93734

The invention relates to a process for the production of a fusion protein, to a gene construct encoding such a fusion protein, and to plasmids useful in the production of fusion proteins.

Fusion proteins with a characteristic C- or N-terminal portion corresponding approximately to the first 100 amino acids of interleukin-2 have already been proposed in the past (DE patent application P35 41 856.7). The interleukin-2 moiety may be derived from mammalian interleukin-2, for example murine or rat interleukin-2, described in EP Application Publication No. 0 091 539 (hereinafter "EP Application Publication"), preferably human interleukin. -2 copies. These fusion proteins are surprisingly stable in the host cell and, due to their low solubility, can be easily distinguished from the host 20's own proteins.

In further developing this idea of the invention, it was surprisingly found that substantially smaller portions of the interleukin-2 molecule are also suitable as a "ballast" portion of such fusion proteins.

The present invention relates to a process for the preparation of fusion proteins having a C- or N-terminal moiety corresponding to an interleukin-2 (IL-2) molecule, preferably a human IL-2 molecule, but containing less than 100 amino acids, with the exception of those portions containing 96 to 99 amino acids by expressing the gene encoding the fusion protein in the host cell. The invention is further defined in the claims. Preferred embodiments are illustrated in more detail below.

Particularly preferably, the synthetic gene corresponding to human-35 interleukin-2 (hereinafter "IL-2"), described in EP-A-0 163 249 and set out in the Annex, is used as starting material. This synthetic gene contains a number of unique restriction cleavage sites that allow the DNA encoding IL-2 to be cleaved into 5 "convenient" segments. - - Sparingly soluble fusion proteins.

The invention also makes it possible to aim for the most advantageous solubility, i.e. good solubility if the product is to be purified chromatographically, for example by means of an antibody column, or low solubility when the host's own soluble solutes are to be obtained in order to carry out pre-purification. proteins, for example by centrifugation.

A particular advantage of the invention is that it is possible to prepare fusion proteins with a relatively small "ballast fraction", since the relative yield of the desired protein is then considerably improved.

A further advantage of the invention is that the "ballast part" can be constructed in such a way that the three-dimensional structure of the desired protein is prevented as little as possible and thus, for example, its folding is not prevented.

• In addition to the desired protein, cleavage of the fusion protein results in a "ballast portion", i.e. an IL-2 derivative. It may have IL-2 activity (T cell proliferation assay) or it may bind to IL-2 receptors. By means of the "building block principle" according to the invention, it is also possible to obtain, as "by-products", IL-2 derivatives in which the biological functions of IL-2 are more or less emphasized.

Particularly suitable embodiments of the invention are illustrated below using the synthetic gene described in EP 0 163 249. This 3 93734 gene is cleaved at the 5 'end by restriction endonuclease EcoRI and at the 3' end by SalI. In addition to the three unique restriction cleavage sites identified by the enzymes PstI, XbaI and SacI used to construct this gene, there are also unique MluI and Pvul cleavage sites at preferred positions. If the sequences between these cleavage sites are denoted by the letters A to F, the synthetic gene can be systematically represented as follows: (EcoRI) -A-PstI-B-MluI-C-XbaI-D-SacI-E-Pvul-F- (SalI).

Segments A to F are thus particularly preferred "building blocks" for a building block system, so in this presentation the "ballast part" of the fusion proteins according to DE patent application P 35 41 856.7 corresponds to segments A 15 to E and the bifunctional protein mentioned in the same application, which contains the entire IL-2 gene. the "ballast portion" segments A to F. In contrast, the gene constructs of the invention involve other combinations of segments A to F, preferably those having less than four of these segments, with segment A encoding the N-terminus of the fusion protein. Optional adapters or linkers may be used, and appropriate adapter or linker sequences may also be added to the C-terminus of the ballast portion, which in this case may also encode amino acids or short amino acid sequences that allow enzymatic expression of the "ballast portion". or chemical removal from or facilitate the desired protein. Of course, the adapter or linker sequences can also be used to make a "ballast portion" custom-made for a particular fusion protein, for example, to provide the desired solubility. Namely, it has surprisingly been shown that the solubility of a fusion protein does not depend on the molecular size, but even relatively small molecules can have a low solubility. Knowing these aspects, which are illustrated in detail in the examples, a person skilled in the art can thus, without laborious experiments, prepare the fusion proteins according to the invention with certain desired properties.

Thus, when the desired protein is a eukaryotic protein, according to the invention, fusion proteins are obtained which consist wholly or practically entirely of eukaryotic protein sequences. However, surprisingly, prokaryotic host cells do not recognize these 10 fusion proteins as foreign proteins, and they are not rapidly degraded by host proteases. Such degradation occurs particularly frequently for host-encoded proteins encoded by cDNA sequences that are to be produced in bacteria. It has now been shown that cDNA sequences 15 can be expressed quite efficiently when "incorporated" into segments according to the invention. To this end, specific vectors can be constructed that contain a polylinker sequence between the sequences with multiple cloning sites for the cDNA sequences. If the cloned cDNA does not contain a stop codon, the cDNA sequence is further protected by a polypeptide encoded by the C-terminal segment.

The cleavage of the fusion protein can be performed chemically or enzymatically in a manner known per se.

‘25 The choice of the appropriate method depends above all on the amino acid sequence of the desired protein. For example, when this does not contain methionine, the binding moiety may be Met, which is chemically cleaved with chlorine or bromocyanine. If the binding moiety has a cysteine at the carboxy terminus or the binding moiety is Cys, enzymatic, cysteine-specific cleavage or chemical cleavage may be performed, for example, after specific S-cyanylation. If the binding moiety at the carboxy terminus is tryptophan or the binding moiety is Trp, chemical cleavage with 35 N-bromosuccimide can be performed.

93734 5

The desired proteins, which do not contain the Asp-Pro pair in their amino acid sequence and are sufficiently acid-fast, can be proteolytically cleaved in a manner known per se as fusion proteins containing this binding moiety.

In this way, proteins containing N-terminal proline or C-terminal aspartic acid are obtained. Thus, modified proteins can also be synthesized in this way.

The Asp-Pro bond can be made even more sensitive to ha-10 when this binding moiety is (Asp) „- Pro or Glu- (Asp)„ - Pro, where n is 1 to 3.

Examples of enzymatic cleavages are also known, in which case modified enzymes with improved specificity can also be used (cf. C. S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proisulin, it is appropriate to select as sequence Y the peptide sequence in which the trypsin-cleavable amino acid (Arg, Lys) is bound to the N-terminal amino acid 20 (Phe) of proinsulin, for example Ala-Ser-Met-Thr-Arg, since then arginine-specific cleavage by trypsin protease can be performed.

If the desired protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, the fusion protein with the appropriate binding moiety can be cleaved by factor Xa (EP-A-0 025 190 and 0 161 973).

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

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

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In bacterial hosts, the promoter and operator are suitably Lac, tae, trp, λ-phage PL or PR / hsp, omp or a synthetic promoter, such as are proposed, for example, in DE-A-34 30 684 (EP-5 0 173 149). Preference is given to a promoter-operator sequence guarantee which is also commercially available (e.g. the expression vector pKK223-3, Pharmacia ("Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, p. 63).

In accordance with the invention, in the case of expression as a fusion protein, it may be appropriate to modify individual triplets of the first amino acids following the ATG start codon to prevent possible base pairing at the mRNA level. Such alterations, as well as alterations, deletions, or additions of individual amino acids in the IL-2 protein moiety, are known to those skilled in the art and are within the scope of the invention. For example, removal of cysteine or replacement of cysteine with other amino acids can be implemented to prevent the formation of undesired disulfide bridges, as described, for example, in EP-A-109 748.

Figures 1-13, like the flow charts, illustrate methods for carrying out the syntheses of Examples 25 denoted by the same numerals. To facilitate an overview, the preparation of starting materials and intermediates is illustrated in Figures A-C. For the sake of clarity, numbering was started in Figures 1-13 from a new decimal number, i.e. Figure 1 (II). The identification numbers of the starting materials which are not the subject of this application end in 0, for example in Figure 2 (20). The figures are not to scale, in particular in the region of the polylinker sequences the scale is suitably stretched. IL-2 sequences are indicated by bold lines, and structural genes corresponding to the desired 35 proteins are otherwise indicated.

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Figure A gives an overview of segments A to F and a combination of segments A and B according to the invention. The starting material is plasmid p159 / 6, the preparation of which is described in detail in EP-A-0 0163 249 and is disclosed in Figure 5 of said publication.

Figure B shows the expression plasmid pEW1000, the preparation of which is described in DE patent application P 3541 856.7 and illustrated in Figure 1. This plasmid is cleaved from the region of the polylinker sequence 10 by appropriate double cleavage to give linearized plasmids (Ex1) - (Ex4).

Figure C shows the preparation of the pUC12 derivative pW226 and the expression plasmid pW226-1, both of which contain segments A and F separated by a polylinker-15 member sequence.

Figure 1 shows the preparation of the pUC12 derivative pKH40 and the expression plasmid pK40, which encode fusion proteins with a protein sequence corresponding to segment A, i.e. the first 22 amino acids of IL-2, followed by the bridging group Thr-Arg followed by proinsulin amino. acid sequence.

Figure 2 shows the construction of plasmid pSL11 and the expression plasmid pSL12, which encode polypeptides in which segment A is followed by a linker corresponding to polysilicon sequences 2 and 20a, followed by the amino acid sequence of proinsulin.

Figure 3 shows the construction of the expression plasmid pK50, which encodes a polypeptide in which segments A and B, i.e. the first 38 amino acids of IL-2, are immediately followed by the amino acid sequence of proinsulin.

Figure 4 shows the construction of the expression plasmid pK51, which encodes a polypeptide in which segments A and B are followed by a bridging group corresponding to sequences 42 and 41, followed by the amino acid sequence of proinsulin.

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Figure 5 shows the construction of the expression plasmid pK52, which differs from pK51 by an added MluI linker 51 encoding an amino acid sequence that allows cleavage by factor Xa. pK52 can also be prepared from pK50 (Figure 3) by digestion with MluI and addition of said MluI linker.

Figure 6 shows the construction of the expression plasmid pK53 from pK51 (Figure 4), which is done in the same way by adding an MluI linker.

Figure 7 shows the construction of the expression plasmid pSL14 from pSL12 (Figure 2) by adding fragment C to the polylinker. Thus, segment C is added immediately as a continuation of segment A. In the next polylinker, the first two amino acids (each Glu) correspond to amino acids 60 and 61 of IL-2. Thus, the IL-2 portion consists of amino acids 1 to 22 and 37 to 61. The remainder of the amino acid sequence corresponds to the sequence encoded by plasmid pSL12 (Figure 2).

Figure 8 shows the construction of the expression plasmid pPH31, which encodes a fusion protein in which segments A to C are followed by the bridging group shown in SEQ ID NO: 81 and followed by the amino acid sequence of proinsulin.

Figure 9 shows the construction of plasmid pK192, which encodes a fusion protein in which segments A '25 and B are followed by methionine followed by the amino acid sequence of hirudin.

Figure 10 shows the construction of plasmid pW214, which encodes a fusion protein in which segments A and B are followed by an amino acid sequence that allows for the cleavage of factor Xa and is followed by "Colony Stimulating Factor" (CSF) of granulocyte macrophages.

Figure 11 shows the construction of the expression plasmid pW233, which encodes a fusion protein in which 35 segments A and C (corresponding to amino acids 1993734-22 and 37-61 of IL-2) are followed by the bridging sequence Leu-Thr-Ile-Asp-Asp-Pro , followed by the amino acid sequence of CSF.

Figure 12 shows the construction of the expression plasmid pX234, which encodes a fusion protein having the following amino acid sequence: segment A (amino acids 1-22) is followed by the bridging group Thr-Arg, segment D (amino acids 59-96 of IL-2), the second bridging sequence Thr -Asp-Asp-Pro and finally CSF.

Figure 13 shows the construction of plasmids pH200 and pH201 as well as the expression plasmid pH202. These plasmids have a polylinker between segments A and F or A, B and F, at which multiple cleavage sites can be cloned with foreign DNA. These plasmids are particularly suitable for cloning cDNA sequences.

The invention is illustrated in more detail in the following examples, the numbering of which corresponds to the numbering of the figures. Unless otherwise stated, percentages are by weight.

Example A

The starting plasmid p159 / 6 is described in EP-A-20 0 163 249 (Figure 5). The sequence defined therein or in the text "DNA sequence I" is divided into segments A to F in Figure A, which are bounded by cleavage sites recognized by the enzymes EcoRI, PstI, MluI, XbaI, SacI, PvuI and SalI. By double cleavage with appropriate enzymes, segments (A) to (F) or also interlocking segments are obtained, for example with EcoRI and MluI the segment (A, B).

Example B

The preparation of expression plasmid pEW1000 is described. 30 (unpublished) in DE patent application P 35 41 856.7 (Figure 1). This plasmid is a derivative of plasmid ptac11 [Amann et al., Gene 25 (1983) 167-178) in which

A synthetic sequence containing a SalI cleavage site is inserted at the EcoRI recognition site. Thus, the expression plasmid pKK177.3 is obtained. Insertion of the Lac rep-1093734 receptor [Farabaugh, Nature 274 (1978) 765-769] provides plasmid pJF118. This is opened from a single Aval restriction site, truncated by about 1000 base pairs in a known manner by treatment with an exonuclease, and lysed. Plasmid pEW1000 is obtained. Cleavage of this plasmid in the polylinker region with EcoRI and HindIII, SalI, PstI or SmaI gives linearized expression plasmids (Ex1), (Ex2), Ex3) and (Ex4).

Example C

The commercial plasmid pUC12 is digested with EcoRI and SalI, and linearized plasmid 1 is isolated. Liga action 1 to segment A, synthetic linker sequence 2 and segment F gives plasmid pW226 3.

E. coli strain 79/02 is transformed in a known manner with the plasmid DNA of the ligation mixture. Cells are seeded in agar plates containing isopropyl-6-D-thio-galactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and 20 μ / ml ampicillin ( Ap). Plasmid DNA, 20 is recovered from the white clones and the formation of plasmid 3 is confirmed by restriction analysis and DNA sequence analysis.

A small EcoRI-HindIII fragment 4 is cleaved from plasmid 3 and isolated. This is ligated into the linearized expression plasmid (Ex1) by a T4 DNA ligase reaction. The resulting plasmid pW226-15 is subjected to restriction analysis.

Competent E. coli strain Mc 1061 cells are transformed with plasmid pW226-1 DNA. Ampicillin-resistant clones are isolated from Aβ-containing agar plates. Plasmid DNA is re-isolated from Mc 1061 cells and re-characterized by restriction analysis. Competent E. coli strain W 3110 cells are transformed with plasmid DNA isolated from E. coli Mc 1061 cells. In the following, E. coli W 3110 cells will be used for all expressions. All expression-35 experiments performed in the given examples were performed under the following conditions.

u 11,93734 An overnight E. coli cell culture containing plasmid 5 is diluted approximately 1: 100 in LB medium (JH Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 50 mg of si-5. / ml ampicillin, and growth is monitored by measuring optical density (OD). When the OD = 0.5, IPTG is added to the culture to a concentration of 1 mmol / l, and the bacteria are separated after 150-180 minutes by centrifugation. The bacteria are boiled for 5 minutes in a buffer mixture (7 mol / l 10 urea, 0.1% SDS, 0.1 mol / l sodium phosphate, pH 7.0), and the samples are transferred to an SDS gel electrophoresis plate. Electrophoresis yields from bacteria containing plasmid 5 a protein band corresponding to the molecular size of the expected protein (6 kilodaltons).

The given induction conditions apply to shake flask cultures; for larger-scale fermentations, appropriately modified OD values and possibly slightly modified IPTG concentrations are appropriate. The resulting protein has no activity in an IL-2-dependent cell proliferation assay (CTLL 2).

Example 1

Plasmid 3 is digested with MluI and SalI, and the two fragments formed are separated by gel electrophoresis. A larger fragment 11 is isolated.

Synthetic oligonucleotide 12 is ligated to blunt-ended DNA encoding proinsulin 13 [Wetekam et al. Gene 19 (1982) 179-183] to give DNA sequence 14.

This is digested with MluI and SalI to give the DNA sequence. This is ligated to fragment 11 to form plasmid pKH40 (16). This is characterized by restriction analysis.

Plasmid 16 is digested with EcoRI and HindIII, and a small fragment 17 is isolated by gel electrophoresis. Ligation of this to the linearized expression plasmid 12 93734 (Ex1) gives the expression plasmid pK40 (18). Expression according to Example C yields a protein that is in the soluble cellular protein fraction after cell disruption. The unchanged proinsulin sequence is determined by the "Wes-5 tern Blot" method.

Example 2 The starting material is plasmid pPH30, which is described in (unpublished) DE patent application P35 41 856.7 in Figure 3c. In the context of the present invention, the IL-2 subsequence is shown in Figure 2 as "A-E" (20). The end of this sequence and the bridging sequence to the proinsulin sequence are shown in Figure 2 as 20a.

Plasmid 20 is digested with Pvul and HindIII, and a small fragment 22 is isolated. In addition, plasmid 15 is digested with EcoRI and Pvul, and a small fragment (23) is isolated. In addition, the vector pUC12 is digested with EcoRI and HindIII, and large fragment 21 is isolated. Ligation of fragments 21, 23 and 22 yields plasmid pSL11 (24). Plasmid 24 is digested with HindIII and partially with EcoRI, 20 and fragment 25 containing segment A and the proinsulin gene is isolated. Ligation to 25 linearized expression plasmids (Ex1) yields the expression plasmid pSL12 (26).

Expression according to Example C and subsequent treatment with fat yields a soluble fusion protein. Western blot analysis with insulin antibodies indicates that this protein contains the entire insulin sequence.

Example 3 .. Amplification of the proinsulin gene is performed using plasmid ptrpED5-1 [Hallewell et al., Gene 9 (1980) 27-47]. This is digested with HindIII and SalI, and a large fragment 31 is isolated. Fragment 31 is ligated to DNA sequence 14 to give plasmid pH106 / 4 (32).

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Plasmid 32 is digested with SalI and MluI, and a small fragment 15 is isolated. The linearized expression plasmid (Ex2), segment (A, B) and fragment 15 are ligated to give expression plasmid pK50 (33).

Expression leading to the encoded fusion protein is made according to Example C. The cells are then separated from the culture broth by centrifugation and disrupted in a "French Press". The protein suspension is separated by centrifugation into soluble and insoluble portions. Each of the 10 fractions is analyzed in a known manner by gel electrophoresis on a 17.5% SDS-polyacrylamide gel and then stained with Coomassie Blue. Surprisingly, it is found that the fusion protein is in the insoluble part. Western blot analysis with insulin antibodies shows that the fusion protein contains the entire proinsulin sequence.

The sediment obtained by the "French Press" violation can be used directly to isolate proinsulin.

Example 4 The starting material is plasmid pPH20 (40), which is described in Figure 3c of DE patent application P 35 41 856.7. Cleavage of this plasmid with EcoRI, complementation of single-stranded ends, and digestion with HindIII yields fragment 41, which shows the DNA sequence of the 40 and 25 parts of interest in this context.

Ligation of the linearized expression plasmid (Ex4) to segment (A, B), synthetic oligonucleotide 42 and fragment 41 yields plasmid pK5i (43).

Example 5 .. Ligation of a linearized expression plasmid (Ex2) to segment (A, B), synthetic oligonucleotide 51 and DNA sequence 15 gives plasmid pK52 (52). The correct orientation of the oligonucleotide 51 is checked by sequence analysis. The plasmid encodes a fusion protein containing the amino acid sequence corresponding to 35 oligonucleotides 51 and is thus cleavable by activated factor Xa.

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Plasmid 52 can also be prepared as follows: Plasmid 33 is cleaved portionwise with MluI and the resulting opened plasmid 53 is ligated to DNA sequence 51, resulting in plasmid pK52.

5 Example 6

When plasmid 43 is partially digested with MluI and the resulting linearized plasmid 61 is ligated to synthetic DNA sequence 51, plasmid pK53 (62) is obtained. This likewise encodes a fusion protein that is cleavable by Akti-10-coated factor Xa. The correct orientation of sequence 51 is confirmed as in Example 5 by DNA sequence analysis.

Example 7

Plasmid 26 is digested with XbaI and partially

Mlulr, and isolating large fragment 71. Ligation to 15 segments (C) yields plasmid pSL14 (72). After expression and cell disruption, the fusion protein is in the soluble cellular protein fraction.

Example 8

Plasmid 20 is digested with XbaI and partially with EcoRI, and complemented by single-stranded ends to give DNA sequence 81. Ligation under "blunt end" conditions yields plasmid pPH31 (82). The fusion protein is in the insoluble cellular protein fraction.

Example 9 Plasmid 90 described in EP-A-0 171 024 (Figure 3) is used as starting material. This plasmid is digested with SalI and then AccI, and a small fragment 91 is isolated. This is ligated to synthetic oligonucleotide 92 to give DNA sequence 93. This digest is digested with MluI to give DNA fragment 94. .

Plasmid 33 is partially digested with MluI and SalI, and a large fragment is isolated. This is ligated to DNA sequence 94 to give the expression plasmid pK192 (96). This encodes a fusion protein in which the first 35 amino acids of IL-2 are followed by methionine and the amino acid sequence of hirudin. The fusion protein is in the soluble cellular protein fraction.

Example 10 The starting material is plasmid pHG23 (100), described in EP-A-0 183 350, which is generally available from the American Type Culture Collection under number ATCC 39000. This plasmid is digested with SfaNI, supplemented with single-stranded ends, reacted with Pstlrn and isolating small fragment 101. 10 Ligation of the linearized expression plasmid (Ex3) to segment (A, B), synthetic oligonucleotide 102 and fragment 101 gives expression plasmid pW214 (103). This plasmid encodes a fusion protein in which the first 38 amino acids of IL-2 are followed by the sequence of oligonucleotide 102, which allows cleavage of the molecule by factor Xa, followed by the amino acid sequence of CSF. The fusion protein is in the insoluble protein fraction after cell disruption.

Example 11 The starting plasmid pW216 (110) is described in DE patent application P 35 45 568.3 (Figure 2b). In this plasmid, the IL-2 sequence corresponding to segments A to E (Pvul cleavage site) is followed by a linker encoding the amino acids Asp-Asp-Pro, followed immediately by the sequence encoding the CSF amino acid sequence. The linker sequence between IL-2 and CSF allows proteolytic cleavage of the fusion protein.

The sequence 111 is isolated from plasmid 110 by digestion with Pvul and HindIII.

Plasmid 3 is digested with MluI and XbaI, and a large fragment 112 is isolated. This is ligated to segment (C) to give plasmid pW227 (113). This plasmid is reacted with EcoRI and HindIII, and the short fragment 114 is isolated. Ligation of this fragment to 35 linearized expression plasmids (Ex1) gives plasmid 16 93734 pW227-1 (115). This plasmid encodes an IL-2 derivative protein that lacks IL-2 activity.

Plasmid 113 is further digested with EcoRI and Pvul, and the short fragment 116 is isolated. Ligation of the linearized expression plasmid (Ex1) to fragments 116 and 111 yields expression plasmid pW233 (117). This encodes an insoluble fusion protein which is proteolytically cleavable thanks to the aforementioned linker.

Example 12 Plasmid 3 is digested with XbaI and SacI, and a large fragment 121 is isolated. Ligation of this to segment (D) gives plasmid pW228 (122). This is digested with EcoRI and HindIII, and a small fragment 123 is isolated. Ligation of the linearized expression plasmid (Ex1) to fragment 123 yields the expression plasmid pW228-1 (124). This plasmid encodes a biologically inactive IL-2 derivative. This plasmid is digested with EcoRI and Pvul, and the short fragment 125 is isolated. Ligation of the linearized expression plasmid (Ex1) to fragments 125 and 111 yields expression plasmid pW234 (126). This encodes a sparingly soluble fusion protein that is likewise proteolytically cleavable.

Example 13

To construct plasmids that are particularly suitable for expressing cDNA sequences, a polylinker sequence 131 is first synthesized.

Ligation of linearized plasmid 1 to segment A, polylinker sequence 131, and segment F yields plasmid ph200 (132).

.. 30 Plasmid 132 is reacted with EcoRI and MluI, and a large fragment 133 is isolated. Ligation of this to segment (A, B) gives plasmid pH201 (134). Plasmid 134 is reacted with EcoRI and HindIII, and a short fragment 135 is isolated. Ligation of this fragment to a linearized expression plasmid (Ex1) gives expression plasmid pH202 (136).

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Plasmid 136 is opened with BamHI, and the cDNA to be expressed is ligated into the linearized plasmid using a commercially available BamHI adapter. According to the orientation of the cDNA, every third sequence is in the same reading frame as (A, B). Unless the cDNA sequence contains a stop codon, the polypeptide sequence encoded by it is further protected by the amino acid sequence corresponding to segment F.

If the cDNA is not in the correct reading frame, the reading frame is transferred, for example, by digesting cDNA-containing plasmids (origin-10 or amplified) with MluI or XbaI (unless the cDNA has cleavage sites corresponding to these enzymes) and filling in single-stranded Klenaseak poly-ends.

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DNA sequence I

Triplet No. 0 1 2

Amino acid Met Ala Pro

Nucleotide No. J_10

5 Coding thread 5 'AA TTC ATG GCG CCG

Uncoded thread ___ 3J_G TAC CGC GGC

(EcoRl) 3 4 5 6 7 8 9 10 1 1 12

Thr Ser Ser Ser Thr lys Lys Thr Gin Leu 10 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 15 Gin Leu Glu His Leu Leu Leu Asp Leu Gin 50 60 70_

CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG

GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC

Pst

20 23 24 25 26 27 28 29 30 31 32

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

ATG ATC CTG AAC GGT ATC AAC AAC TAC AAA

TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT

- 25 33 34 35 36 37 38 39 40 41 42

Asn Pro Lys Leu Thr Are Met Leu Thr Phe 110 120 130

AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC

30 TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG

MluI

II

19 93734 43 44 45 46 47 48 49 50 51 52

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

AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA

5 TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT

53 54 55 56 57 58 59 60 61 62

Leu Lys His Leu Gin Cys Leu Glu Glu Glu 170 180 _ 190

10 CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG

GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC

Xbal 63 64 65 66 67 68 69 70 71 72

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu 15 200 210 220

CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG

GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC

73 74 75 76 77 78 79 80 81 82 20 Ala Gin Ser Lys Asn Phe His Leu Are Pro 230 240 250

GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG

CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC

25 83 84 85 86 87 88 89 90 91 92

Are Asp Leu lie Ser Asn lie Asn Val lie 260 270 280

CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC

GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG

'30 93 94 95 96 97 98 99 100 101 102

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

GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG

35 CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC

Sac

20 93734 103 104 105 106 107 10B 109 110 111 112

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

TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG

5 AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC

113 114 115 116 117 118 119 120 121 122

Thr Ile Val Glu Phe Leu Asn Are Trp lie _350 360 370

10 ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC

TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG

Pvul 123 124 125 126 127 12Θ 129 130 131 132

Thr Phe Cys Gin Ser lie lie Ser Thr Leu 15 380 390 400

ACC TTC TGC CAG TCG ATC ATC TCT ACC CTG

TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC

133 134 135 20 Thr 410__ ACC TGA TAG 3 'TGG ACT ATC AGC T 5' (Sail) tt

Claims (9)

A process for the initial lining of fusion proteins with a C- or N-terminal moiety corresponding to an interleukin-2 (IL-2) molecule, preferably human IL-2, but containing less than 100 amino acids, with except for the moieties containing 96 - 99 amino acids, characterized in that a gene encoding the fusion protein is expressed in a host cell. 10 2. A method according to claim 1, characterized in that the gene encoding the IL-2 moiety comprises part of the DNA sequence I (appendix). Method according to claim 2, characterized in that the gene encoding the IL-2 portion consists of one, two or three of segments A - F of the IL-2 gene (EcoRI) -A-Pstl-B. -Mulul-C-Xbal-D-Sacl-E-Pvul-F- (Sali) in selectable order and possibly interconnected by means of adapter or switching sequences. 4. A method according to any one of claims 1-3, characterized in that between the IL-2 sequence and the amino acid sequence of the desired protein is an amino acid or amino acid sequence which allows chemical or enzymatic cleavage of the desired protein from IL-2. the moiety, preferably the amino acid Met, Cys, Trp, Lys or Arg, or an amino acid sequence containing some (s) of these amino acids in the C-terminus. 5. A method according to claim 4, characterized in that the amino acid sequence is Asp-Pro or Ile-Glu-Gly-Arg or contains this sequence in C-. 30 terminal. Method according to any of claims 1-5, characterized in that the gene encoding the fusion protein is expressed in a bacterial cell, preferably in E. coli. 93734 Use of fusion protein soin may be obtained according to any of claims 1-6 for the production of desired proteins. A gene structure, characterized in that it encodes a fusion protein obtainable according to any one of claims 1-6. 9. pUC derivatives pW226 (Figure C), pW227 (Figure 11), pW228 (Figure 12), pH 200 and pH 201 (Figure 13); the expression plasmids pW226-1 (Figure C), pW227-1 (Figure 11), pW228-1 (Figure 12) and pH 202 (Figure 13); expression plasmids encoding a fusion protein containing an IL-2 moiety as claimed in patent claim 1, a bridge moiety and proinsulin: pK40 (Figure 1), pSL12 (Figure 2), pK50 (Figure 3), pK51 (Figure 4) , pK52 (Figure 5), pK53 (Figure 6), pSL14 (Figure 7), 15 pPH31 (Figure 8); an expression plasmid encoding a fusion protein containing an IL-2 moiety as claimed in claim 1, a bridge moiety and hirudin: pK192 (Figure 9); expression plasmids encoding a fusion protein containing an IL-2 moiety as claimed in claim 1, a bridge moiety and GM-CSF: pW214 (Figure 10), pW233 (Figure 11), pW234 (Figure 12).