IE59779B1 - Gm-csf protein, its derivatives, the preparation of proteins of this type, and their use - Google Patents

Abstract

CSF proteins which are biologically active are obtained by expression of a gene coding for human granulocyte macrophage colony stimulating factor (CSF) in bacteria. Biologically active derivatives with a modified amino acid sequence are obtained by altering the natural or synthetic gene structure.

Description

Human granulocyte macrophage colony-stimulating factor (GM-CSF) is a glycoprotein with a molecular weight of about 23,,000 dalton. The cDNA sequence and the expression of th® glycoprotein in mammalian cells have already been 5 disclosed (G.G. >?ong et al.,. Science 228 (1985) ,. 810-815, D. Metcalf, Science 229 (1985), 15-22).

The invention relates to human granulocyte macrophage colony-stimulating factor proteins (GM-CSF) of the formula Pro- (As) s-CSF (12-125)-2 in which (As)s denotes call or some of th® first 11 siaino acids of th® natural GM-CSF sequence, and 2 denotes Glu or Asp, hereinafter referred to as CSF. Further aspects of the invention and preferred embodiments thereof are illustrated in further detail below or are defined in the patent claims.

The invention furthermore relates to the preparation of CSF by expression in bacteria, in particular in S. coli. In particular, it is possible to use for this purpose the published cDNA sequences which can be obtained in a manner known per se, preferably by synthesis.

The invention additionally relates to expression vectors for use in bacteria, in particular in S. coli, which contain, in a suitable arrangement ("operatively linked to), a DNA coding for CSF or a CSF fusion protein.

The invention additionally relates fco biologically active derivatives of CSF which can be obtained by modifications, which are known per se, of the DNA sequences. Thus, for example, it is possible in the construction of vectors for fusion proteins to incorporate cleavage sites which, after elimination of the CSF protein, have C-terminal and N-texminal modifications in the amino acid sequence. Furthermore, the invention relates to the use of proteins of this typ® In medical treatment and to their use for the preparation of medicaments, and to medicaments which contain CSF protein and its biologically active derivatives e in particular medicaments for the stimulation of proliferation of .^emopoietic cells and for promotion ox the formation of granulocytes and macrophages.

Th® invention is furthermore illustrated by Figures 1 to 15, each of which explains, mostly in the form of a flow diagram, the processes of the examples of th© same numbers. These figures ar® not to scale, in particular th® scale has been expanded in the region of the polylinkers.

Thus, Figure 1 and its continuations la and lb show the preparation of the vector pw 225 which is used for the direct expression of (Met-)CSF. This product can be converted by acid cleavage into CSF according to the invention. The figures which follow relate to vectors which result in the expression of fusion proteins in which a ballast' protein, which Is derived from a partsequence of human, interleukin-2, hereinafter IL-2 or aIL-2, is located at the Η-terminal end in front of the CSF amino’'acid sequence: Figure 2 and its continuations 2a and 2b show the preparation of the vector pw 216 which codes for a fusion protein from which is obtained, by acid cleavage, a CSF derivative which Is extended at the N-terminal end by the amino acid proline.

Figure 3 shows the synthesis of the vector pH 240 which codes for a fusion protein which results, after acid cleavage, in a CSF derivative which has proline in place of the first amino acid (alanine).

Figure 4 relates to the preparation of the vector prar 241 which codes for a fusion protein which results, after acid cleavage, ia a CSF derivative in which the first amino acid (alanine) is missing.

Figure 5 demonstrates the preparation of the vector pW 242 which codes for a fusion protein which results, after acid cleavage, in a CSF derivative in which the first five amino's acids have been eliminated.

Figure 6 relates to th© preparation of the vector pw 243 which codes for a fusion protein which results, after acid cleavage, in a CSF derivative in which the first seven amino acids are missing.

Figure 7 shows the synthesis of the vector pW 244 which codes for a fusion protein with which is obtained, after acid cleavage, a CSF derivative in which, the first 11 amino acids have been eliminated.

Figure 8 and Its continuation 8a show the synthesis of the vector pW 246. This codes for a fusion protein In which two modified sequences, denoted CSF', follow the IL-2 part-sequence. Acid cleavage results la a CSF derivative in which proline is located at the N-terminal end JLn front of the first amino acid proline and in which the last amino acid has been replaced by aspartic acid.

Figure 9 shows the synthesis of the vector pW 247 which codes for a fusion protein In which three CSF’ sequences follow the IL-2 part-sequence. Acid cleavage results in the CSF derivative characterized in Figure 8 being obtained.

Figure 10 and its continuation Figure 10a show the preparation of th® hybrid plasmids pS 200 to 204 which contain synthetic CSF DMA part-sequences, the plasmid pS 200 containing synthesis block ϊ-, shown in Appendix I, plasmid pS 201 containing synthesis block II shown in Appendix II, plasmid pS 202 containing synthesis block III shown in Appendix III, plasmid pS 203 containing the entire synthetic gene, and ps 204 representing an expression plasmid which likewise contains th® entire synthetic CSF DMA sequence. Expression and acid cleavage result in the same CSF derivative as described in Figure 2 being obtainedFigure 11 and its continuation Figure Ila show the synthesis 'Of the expression plasmid pS 207 which codes for a fusion protein which provides, after cleavage with N-bromosuccinimide, a CSF derivative in which Trp in each of positions 13 and 122 has been replaced by His.

Figure 12 shows a synthetic DNA part-sequence which permits the preparation of a CSF derivative in which lie in position 100 has been replaced by Thr.

Figure 13 and its continuation Figure 13a show the synthesis of the expression plasmid pS 210 which codes for a fusion protein which provides, after cleavage with cyanogen bromide, a CSF derivative in which all methionine residues have been replaced by neutral amino acids, namely by lie in position 36 and by Leu in positions 46, and 80.

Figure 14 ^shows a synthetic DNA sequence which permits, in accordance with the synthesis scheme in Figure 13, the preparation of a CSF derivative in which Met in position 36 has been replaced by lie, and Met in position 46 has bees replaced, by Leu, and a single Leu residue is present in place of amino acids 79 and 30» Finally, Figure 15 shows a synthetic DNA whose use in the synthesis scheme shown in Figure 13 permits the prepara25 tion of a CSF derivative in which Met in position 36 has been replaced by lie and in position 46 has been replaced by Leu, and in which the two amino acids in positions 79 and 30 have been deleted.

The possible variations explained in these figures and examples are, of course, merely examples of the large numbers of modifications which are possible'according to the invention. Thus, it is also possible in a manner known per ss t© use other protein sequences, especially bacterial, as the ballast" portion of the fusion proteins, and it is possible to use all customary methods for the linkage and cleavage of the fusion proteinsι it being possible for other CSP derivatives with a modified amino acid sequence in the molecule or at both ends of the molecule to result. The choice of the IL-2 sequence and the synthetic DHA sequences and the cleavage of the fusion proteins should thus be viewed merely as preferred embodiments of the invention which can be varied in a manner known per se.

It has emerged that the open reading frame ''.comprising a DHA which codes for interleukin-2 is particularly advantageous as an expression aid for the expression of peptides and proteins, and that an N-terminal portion of IL-2 which essentially corresponds to the first 100 amino acids is particularly well suited for the preparation of fusion proteins. The primary product obtained in this way is a fusion protein which is composed entirely or very predominantly of eukaryotic protein sequences. Surprisingly, this protein is apparently not recognized as being a foreign protein by the proteases which are intrinsic to the host, nor is It immediately degraded again- Another advantage Is that the fusion proteins according to the invention ar® sparingly soluble or insoluble and thus can easily be removed, appropriately by centrifugation, from -the soluble proteins.

Since, according to the invention, the functioning of the "ballast portion of the fusion protein does not depend on the IL-2 portion, being a biologically active molecule, it likewise does not depend on the exact structure of the IL-2 portion* It suffices for this purpose that essentially the first 100 N-terminal amino acids are present. Thus, it is possible, for example, to carry out at the H-terminal end modifications which permit cleavage of the fusion protein In the case where the desired protein Is located N-terminal thereto. Conversely, modifications at the C-terminal end can be carried out in order to permit or facilitate the elimination of the desired protein.

The natural DNA sequence coding for human IL-2 is disclosed in the Buropean Patent Application with the publication number 0,091,539. The literature quoted there also relates to mouse and rat IL-2. These mammalian DNAs can b® used for the synthesis of the proteins according to tha invention. However, it is more appropriate to start from a synthetic DNA, particularly advantageously from the DNA for human IL-2 which has been described in German Offenlegungsschrift 3,419,995 and in Patent Specification No. 1319/85. This synthetic DNA not only has the advantage that in its choice of codons it is suited to the circumstances in the host which is used most frequently, Ξ. coli, but it also contains a number of cleavage sites for restriction endonucleases at the start and in the region of the 100th triplet, it being possible to make use of these according to the invention. However, this does not rule out modifications to the DNA being carried out in the region lying between them, it being possible to make use of the other cleavage sites.

If use is mad® of the nucleases Ban II, Sac I or Sst I, then the IL-2 part-sequence which is obtained codes for about 95 amino acids. This length is, in general, sufficient to obtain an insoluble fusion protein. If the lack of solubility is still inadequate, for example in the case of a desired hydrophilic CSF derivative, but it is not wanted to make use of cleavage sites located nearer to the C-terminal end - in order to produce as little ’•ballast8' as possible - , then the DNA sequence can be extended at the N-terminal and/or C-terminal end by appropriate adapters or linkers and thus the ballastportion can be ’’tailored’ to requirements. Of course, it is also possible to use th© DNA sequence - more or less up to the end and thus generate biologically active IL-2 - modified where appropriate - as by-product®.

Th© cleavage of the fusion protein can be carried out chemically or enzymatically in a manner known per se. The choice of the suitable method dependst in particulars oa the amino acid sequence of the desired protein- If there is tryptophan or methionine at the carboxyl terminal end of the bridge member or if Y represents Trp or Met, then chemical cleavage with N-bromosuccinimide or cyanogen halide can be carried out in the cases where the particular CSF derivatives which are synthesized do not contain these amino acids.

CSF and those of its derivatives which contain in their amino acid sequence Asp - Pro and are sufficiently stable to acid can, as already shown above, be cleaved proteolytically in a manner known per se. This results in proteins which contain proline at the ^-terminal end or aspartic acid at the C-terminal end being obtained. Thus, it is possible in this way also to synthesize modified proteins.

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

-Examples for, enzymatic cleavages are likewise known, it also being possible to use modified enzymes having improved specificity (cf- C.S- Craik et al., Science 228 (1985) 291-297).

Th® fusion protein is obtained by expression in a bacterial expression system in a manner known per se. Suitable for this purpose are all known host-vector systems, such as bacteria o£ the varieties Streptomyces, 3. subtilis, Salmonella typhimurium or Serratia marcescens, in particular 3. coli.

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

It is appropriate tor this to select the promoter and operator from the group comprising trp, lac, tac, PLor Pa of phage λ, hsp, oisp or a synthetic promoter as proposed in, for example, German Offenlegungsscrift 3,430,583 or Patent Specification No. 2048/85,, The tac promoter-operator sequence is advantageous, and this is now commercially available (for example expression vector pRX223-3, Fharmacia, Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, page 63).

It may prove to be appropriate in the expression of the fusion protein according to the invention to modify individual triplets for the first few amino acids after the ATG start codon in order to prevent any base-pairing at the level of the mRNA. Modifications of this type, such as deletions or additions of individual amino acids, are familiar to the expert, and the invention likewise relates tq them.

Particularly advantageous CSF derivatives are those containing N-texminal proline, since proteins of this type are more stable to attack by proteases. The CSF derivative which has the entire CSF amino acid sequence following the proline added to the N-terminal end is particularly preferred. However, it has emerged, surprisingly, that the variants of the CSF molecule obtained by elimination of the first 11 amino acids also have biological activity.

Variants of the invention which are also advantageous are those which initially result in fusion proteins which contain the CSF sequence more than once, advantageously twice or three times. 3y their nature, the ballast portion in these fusion proteins is reduced, and thus the yield of the desired protein is increased. ίο The plasmid pHG 23 which was obtained by incorporation of the CSF cDNA sequence into the Pst I cleavage site of BR 322 has been deposited, in B. coli, at th© American Type Culture Collection under nuiabex ATCC 39S00. The DNA sequence of this corresponds to the variant described ia Figure 3 (3) of wohg et al. The incorporation made use of the Pst I cleavage site near the 5* end, on the one hand, and of a Pst I site introduced at the 3' end by GC tailing (EP-A 0,183,350)..

Example 1 Direct Expression of CSF The commercially available vector pUC 12 is opened with the restriction enzymes Sma I and Pst I, and the large fragment (1) is isolated.

By cutting the cDNA sequence for CSF with the enzymes Sfa NI and Pst I is obtained the fragment (2) which is ligated, with the synthetic linker (3) and then with the pUC 12 fragment (1). The hybrid plasmid pW 201 (4) which is thus obtained contains the CSF DNA sequence following the start codon ATGThe hybrid plasmid (4) is opened with Neo I, and the protruding ends ar© filled in to give the blunt-ended fragment (5) . The vector pUC 12 is opened with th© enzyme Eco RI, whereupon the protruding end® ar© filled in. This is followed by treatment with bovin® alkaline phosphatase, the pUC 12 derivative (6) being obtained.

Ligation of the fragments (5) and (6) results in vectors which contain the CSF DNA sequence ia both orientations being obtained. They are called pW 203 (7).

Using Eco RI and R®a I on the vector (7) results in isolation of th© fragment (8) which contains the codons for amino acids S3 to 127 of CSF. On the other hand, cutting ths vector (4) with Neo I and Rea I results ia isolation of th© fragment (9) which contains ths codons for amino acids 1 to 61 of CSF.

The plasmid pH 131/5 (German Offenlegungsschrift 3,514,113 or Patent Specification No. 1031/86, Example 1, Figure 1) (10) is cut with Pvu II, the small fragment is removed, and the larger one is ligated to give th® plasmid pPH 160 (11) which is present in E. coli cells in a higher copy number than, pH 131/5. The plasmid (11) is opened with Neo I and Eco RI, and the large fragment (12) is isolated.

Th® fragments (8,, (9) and (12) ar© now ligated to give the hybrid plasmid pH 206 (13). This restores the codon for amino acid 62.

Th© commercially available plasmid pKK 65-10 (PL Biochemical Inc.) is cleaved with Eco RI, and the fragment (14) which contains the two terminators Tl and T2 is isolated. This fragment (14) is inserted into the plasmid (13)^ which has been opened with Eco RI, the plasmid pN f 225 (15) being obtained.

E. coli 24 bacteria which contain the plasmid (15) are cultured in LB medium (J-B. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 30 to 50 /«g/ml ampicillin at 37®C overnight.

Ths culture is diluted ia the ratio 1:100 with M9 medium (J.M- Miller, op. cit.) which contains 200 /na/I casamino acids and 1 /»g/l thiamine, and the mixture is incubated at' 37*C with continuous agitation. At an ΟΟβοο β θ·5 or indolyl-3-acrylic acid Is added to a final concentration of 15 /«g/l, and the mixture is incubated for 2 to 3 hours or 16 hours respectively. The bacteria are then removed by centrifugation. The bacteria are boiled for five minutes in a buffer mixture (7M urea, 0.1% SDS, 0.1 M sodium phosphate, pH 7.0), and samples are applied to an SDS gel electrophoresis plate. It emerges that the protein pattern of cells whose trp operon has been induced contains a new protein, in the range of about 14,000-18,000 dalton, which ie not found with son-induced cells.

The induction conditions which have been indicated apply 5 to shake cultures; for larger fermentations appropriately modified OD values and, where appropriate, slight variations in the inducer concentrations are advantageous.

Example 2 Pro°-CSF The vector pOC 12 is opened with Eco RI and Pst I, and the large fragment (16) is isolated. This fragment (16) is ligated with the synthetic DNA fragment (17) and the fragment (2) (Example 1; Figure 1). Competent cells of E. coli JM 103 are transformed with the ligation mixture, and the desired clones which contain the plasmid pN 212 (18) are selected.

Ths fragment (19) which contains the CSF sequence is cut out of the plasmid DNA using Pvu I and Pst I.

Insertion of the lac repressor (P-J. Farabaugh, Nature 274 (1978) 765-769) into the plasmid pKK 177-3 contain the pue 8 polylinker (Amann et al., Gene 25 (1983) 167; Patent Specification No. 1930/84) results in the plasmid pJF 118 (20) being obtained (Fig. 2a; cf. German Patent Application P 35 26 995.2, Example 6, Fig. 6). The latter is opened at the unique restriction site for Ava I, and is reduced in size by about 1,000 bp by exonuclease treatment in a manner known per se. Ligation results in the plasmid p2B 1000 (21) being obtained, in which the lac repressor gene is completely retained but which, because of the reduction in size, is present in a markedly higher copy number than the initial plasmid.

In place of the plasmid pKK 177-3, it- is also possible to start from the abovementioned commercially available plasmid pKK 223-3, to incorporate the lac repressor, and to shorten the resulting product analogously.

The plasmid pEW 1000 (21) is opened with the restriction enzymes EcoR I and Sal I, and the fragment (22) is isolated.

The plasmid pl59/6 (23), prepared as described in German Offenlegungsschrift 3,419,995 (Patent Specification No. 1319/85), Exanple 4 (figure 5), is opened with the restriction enzymes Eco RI and SAI I, and the small fragment (24), which contains the IL-2 sequence, is isolated.

The hybrid plasmid pEW 1001 (25) is obtained by ligation of the fragments (22) and (24).

On the one hand, the plasmid (25) is opened with Eco RI and Pvu I, the fragment (26) which contains the largest part of the IL-2 sequence being obtained.. This partsequence is denoted nlL2"’ in the figures.

On the other hand, the plasmid (25) is opened with Eco RI and Pst I, and the large fragment (27) is isolated.

Ligation of th© fragments (19,, (26, and (27,, transformation of competent S. coli 294 cells, and selection results in clones which contain the plasmid pW 216 (28) being obtained. The plasmid DMA is characterized by restriction analysis and DNA sequence analysis.

An overnight culture of S. coli cells which contain the plasmid (23) is diluted with LB medium (J. H- Miller, opcit-), which contains 50 pg/ml ampicillin, in the ratio of about 1:100, and the growth is followed via measurement of the OD. At OD = 0.5, the culture is adjusted to 1 mM in isopropyl ^-galactopyranoside (IPTG) and, after 150 to 180 minutes, the bacteria are removed by centrifugation. The bacteria are boiled for five minutes xn a buffer mixture (7M urea, 0.1% SDS, 0.1 M sodium phosphate, pS 7.0), and samples are applied to an SDS gel electrophoresis plate. Following electrophoresis, a protein band which ' corresponds to the size of th© expected fusion protein is obtained from bacteria which contain the plasmid (28). After disruption of th® bacteria (French press; *Dyno mill) and. centrifugation, the fusion protein is located in the sediment so that it is possible already to remove considerable amounts of the other proteins with the supernatant. Isolation of the fusion protein, is followed by acid cleavage to liberate the expected CSF derivative which contains an additional N-terminal proline. This shows activity in the biological test.

The induction conditions which have been indicated apply to shake cultures; for larger fermentations appropriately modified OD values and, where appropriate, slight variations in the IPTG concentrations are advantageousExample 3 ProJ-CSF (2-127, Ligation of the fragments (2) (Figure 1, and (16) (Figure 2) with the synthetic DNA sequence (29, results in the hybrid plasmid (30) which corresponds to the plasmid (18) apart from the synthetic DNA sequence.

Pvu I and Pst I are used to cut out of the plasmid (30) the fragment (31) which contains the CSF DNA sequence in which, however, the codon for the first amino acid has been replaced by a codon for proline. Ligation of the fragment (31) with the fragments ¢26) and (27) results in the hybrid plasmid gffl 240 (32) being obtained. Expression in S. coli, which is carried out as in Bxample 2, provides & CSF derivative in which the first amino acid has been replaced by proline. This derivative also shows biological activity.

* Trade Mark Example .4 CSF (2-127) A plasmid which contains the CSF DNA sequence with a Pst I restriction site at its 3' end, for example the plasmid pHG 23 (ATCC 39900), is cleaved with Sfa NI, and the linearized plasmid (34) is partially filled in using Klenow polymerase and GTP- The protruding nucleotide A is eliminated using SI nuclease, and then the fragment (35) is cut out with Pst I.

Ligation of th© fragment (35) with the synthetic DNA sequence (36) and the fragment (16) (Figure 2) results in the plasmid (37), which is analogous to plasmid (18), being obtained.

Pvu I and Pst I are used to cut the fragment (38) out of the plasmid (37). This fragment is ligated with the fragments (26) and (27), by which means the plasmid pw 241 (39) is obtained.

Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSP derivative missing ths first amino acid. This derivative is biologically active.

Example 5 CSF (6-127) The plasmid (33) (or a corresponding plasmid which contains the· CSF DNA sequence) is first totally cleaved with Pst I and then partially cleaved with Sst NI,. and the fragment (40) is isolated.

The synthetic DNA sequences (41) and (36) (Figure 4) are first ligated to give the sequence (42), and the latter is then ligated with the fragment (16) (Figure 2) and the fragment (40), the plasmid pW 212 (43) being obtained.

Pvu I and Pst I are used to isolate from the plasmid (43) the fragment (44, which contains the DMA sequence for the CSF derivative. This fragment (44, is ligated with the fragments (26) and (27,t which results in the hybrid plasmid pW 242 (45).

Expression as in Examples 2 results in a fusion protein from which is obtained, after acid cleavage, a CSF derivative missing the first five amino acids. This product is also biologically active.

Example 6 CSF (8-127) When first the synthetic DNA sequence (36) (Figure 4) is ligated with the synthetic DMA sequence (46), and there15 after the resulting DMA fragment (47) is ligated with the fragments (40) and (16), then the hybrid plasmid (48) is obtained. Pvu I and Pst I are used to cut out of the latter the fragment (49) which contains the DMA sequence for the CSF derivative. Ligation of the fragments (49), (26) and (27) provides the hybrid plasmid pW 243 (50) which corresponds to the plasmid (45) apart from the shortened DMA sequence for th® CSF derivative·* Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSF derivative missing th® first seven amino acids. This derivative is also biologically active.

Example 7_ CSF (12-127) When ths synthetic DMA sequence (51) is ligated with the fragment© (33) and (16) then th® hybrid plasmid (52) is obtained. When Pvu I and Pst I are used to cut out of the latter the sequence (53), which contains the DNA sequence for the CSF derivative, and this fragment (53) is ligated with the fragments (26) and (27) then the hybrid plasmid pw 244 (54) which corresponds to the plasmid (45) apart from the shortened CSF sequence is obtained.

Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSF derivative from which amino acids 1 to 11 have been, eliminated. This shortened molecule is also biologically active.

Example 8 Pro°~CSF (1-126) -Asp The DNA sequence (19) (Figure 2) is partially cleaved with Bst NI, and the fragment (55), which contains the largest part of the CSF sequence, is isolated.

Cleavage of the plasmid (33) (figure 4) (or of a corresponding plasmid, which contains the CSF DNA sequence) first with Pst I and then partially with Bst NI results in the DMA sequence (56) which comprises th© largest part of the CSF sequence being obtainedThe DNA sequence (57) is synthesized which together with the sequence (56) provides a DNA sequence which codes for a CSF derivative in which the C-terminal glutamic acid has been replaced by aspartic acid.

The vector pUC 13 is opened with Pst I and Sma I, and the large fragment (58) is isolated. When this linearized plasmid (58) Is ligated with the fragments (56) and (57),, then the hybrid plasmid pH 245 (59) with the modification of the C-terminal sequence is obtained.

Sfa NI and Pst I are used to cut out of the plasmid (59) the fragment (SO) which contains the modified CSF DNA sequence. This fragment (SO) is ligated with the synthetic DNA sequence (SI) and. the fragment (55), the DNA sequence (62) being obtained. The latter is ligated with the DNA fragments -(26) -and (27) (Figure 2), the hybrid plasmid pW 246 (S3) being obtained. This plasmid is shown twice in Figure 8a, the lower representation indicating the amino acid sequence of the coded fusion protein.

Expression as in Example 2 results in a fusion protein from which, after acid cleavage, is derived a CSF deriva10 tive which is extended by an N-texminal proline and in which, additionally, the final amino acid has been replaced by aspartic acid. This derivative is biologically active.

Example 9 Pro°-CSF(1-126)-Asp The hybrid plasmid (63) (Figure 3) is cleaved with Eco RI and Pst I,, and the fragment which contains th© two modified CSF sequences following the IL-2 part-sequence is isolated. This sequence (64) is partially cleaved with Rsa I, and the two fragments (65) and (66) are isolated.

The fragment (66) is cleared with Bat NI, and the fragment (67) is isolated. Ligation of the DNA sequences (27), (65), (67), (61) and (60) results in the hybrid plasmid pW 247 (68) ia which the ligated sequences are arranged in the .specified sequence.

Expression as in Example 2 provides a fusion protein from which results, after acid cleavage, the same CSF derivative ae in Sxample 8.

Sxample_10 Synthetic gene (for Fro°-CSF) Processes known per se, for example the phosphite method (German Offenlegungsschriften 3,327,007, 3,328,793, 3,409,966, 3,414,831 and 3,419,995) are used to synthesize the three synthesis blocks I (GSF-ϊ), designated (69) in th© figures, II (CSF-1I), (70) in the figures, and III (CSF-ϊϊϊ), (71) in the figures. The synthesised oligonucleotides la to Im, XIa to Ilf and Ilia to 1111 are indicated in the nucleotide sequence of these synthesis blocks (Appendix)» The choice of the nucleotides for the synthetic gene 10 entailed provision not only of unique cleavage sites at the points of union of the three synthesis blocks but also of a number of unique restriction sites inside the gene fragments. These are listed in the tables below.

These unique restriction sites can be used, In a manner 15 known per se, to exchange, add, or delete codons for amino acids.

Synthesis Block I (CSP I) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) Bar I GGWGCC 1 Hpa II C 4CGG 4 Has II GGCGC *C 4 fee I GCC 4GGC 5 ?vu I CGATCG 13 Sal I G4TCGAC 24 Acc I GT CGAC 25 Sine II GTG *0A0 26 Spa 1/ GTT4AAC 48 Sine II Hha I GCGC 66 Hinf I S+AGTC 88 Bra I TCG4C5A 89 Xma III C -SGGCCG 95 Sac 11 CCGC4CG sco av GAT «ATC 128 Synthesis Block II fCSF-IIl Enzyme Recognition sequence Cut after nucleotide no. (coding strand) Afl III A4CATGT 157 Mlu 1 A-CGCGT 169 Xho 1 G»TOGAC 175 Tag I T4CGA 176 Bga V Mta> GACGC (5/10) 177 Ava T C*TC6AC4 177 Alu I AG4CT 180 Sec 1/ GAGCT4C 182 3gi AT Stu 1/ AGG CCT 194 Bae I Synthesis Block III (CSF-III) Ensysae Recognition sequence Cut after nucleotide no. (coding strand) Ail II C4TTAAG 217 Sa© III GGCC 224 A pa I GGGCOC 227 Hal I CCTC (7/7) 23S She I 64CIAGC ,241 Hae I C4TAG 242 Aha IT GA CGTCe· 280 Aat II GACGT4C 283 Sci 11 G4CGC 287 Hat I TCC44GCA 288 San 3AI/ 4GATC 296 Mho I Dpn I GA*IC 298 Asu II TTCGAA 308 Aha III TTTUAA 318 Ava II G4GTCC 382 Eco RII CCAGG 384 Sst 11/ CC4AGG 380 Scr El The three synthesis blocks were first individually cloned, amplified in S. coli and re-isolated: Synthesis block CSF-I (SS) is incorporated in th© pUC 12 derivative (15)z the plasmid pS 200 (72) being obtainedpUC 12 is opened with the restriction enzymes Pst I and Hind III and, the linearized plasmid ¢73) is ligated with synthesis block CSF-II (70), the plasmid pS 201 (74) being obtainedpUC 13 is opened with Sind III and Sraa I, and the linearised plasmid (75) is ligated with CSF-III (71), th© plasmid pS 202 (76) being obtained.

The re-isolated synthesis blocks (69), (70) and (71) are now ligated in the vector puC 12 (77) which has been linearised with Eco Rl and Sma I, the result being the plasmid pS 203 (78). This hybrid plasmid is - as the plasmids with the individual synthesis blocks - amplified in E. coli 79/02,, and the synthetic gene is characterized by restriction analysis and sequence analysis.

The plasmid (78) is cleaved with Pvu I partially and with 10 Bam HI, and the small fragment (79) with the complete CSF sequence is isolated..

The expression plasmid (21) is opened with Eco RI and Bam HI, and the large fragment (80) is isolated. This fragment (80) is now ligated with the fragment (26) which contains the IL-2 part-sequence and the synthetic gene (79). This results in the plasmid pS 204 (81) which codes for a fusion protein in which the IL-2 part-sequence is followed first by th® bridge member which permits acid cleavage and then by the amino acid sequence of CSF.

Thus, acid cleavage results in a CSF derivative which is extended by proline at th© N-terminal end.

Example 11 CSF(1-12)His(14-121,His(123-127) When the nucleotides in synthesis block I up to So. 48 (cleavage site for Spa 1) are replaced by the synthetic sequences (82, and (83), then the result is a modified synthesis block I which codes for a CSF I analog xn which there is Trp io, front of the first amino acid (Ala), and Trp in position 13 has been replaced by Sis.

The plasmid (72) (Figure 10) is opened with Eco RI and Spa I, and the large fragment (84) is isolated. The latter is now ligated with the synthetic fragments (82) and (83), the plasmid pS 205 (85) which codes for this modified CSP I (C5FI) being obtainedThe plasmijd (76) (Figure 10) is opened with Bind III and Sal I, and the small (86) and large (87) fragments are isolated. The small fragment (86) is then cut with Tag I, and the fragment (88) is isolatedThe large fragment (87) is now ligated with (88) and with the synthetic fragment (89) in which the codon for Trp in position 122 has been, replaced by His, the plasmid pS 206 (90) which codes for the modified CSF III (CSF III') being obtained- This plasmid is transformed into Ξ- coli, amplified, re-isolated, cut with Hind III and Sal I, and the small fragment (91) which codes for CSF III' is isolated.

The plasmid (85) is cut with Pvu I partially and with Pst I, and the small fragment (92) which codes for CSF 1' is isolatedWhen the fragments (22), (26), (92), (70) and (91) are now ligated then the plasmid Ps 207 (93) is obtained.

This codes for a fusion protein in which the IL-2 partsequence is followed by a bridge member which contains Trp immediately in front of the first amino acid of CSF .(Ala) - Since Trp In positions 13 and 122 of the CSF molecule have been replaced by Bis, it is now possible to cleave the fusion protein with N-bromosuccinimide. This results in the CSF derivative In which tryptophan in both positions has been replaced by histidine24 Sxamgle_12 CSF(1-99)Thr(101-127) When, in the synthesis ox the synthesis block III, oligonucleotides Tile and Illf are replaced by the synthetic sequence (94) and the process is otherwise carried out as in Example 10, then a CSF derivative in which lie in position 100 has been replaced by Thr is obtained.

Example 13 CSF(1-35)lie(37-45)Leu(47-78)Leu-Leu(81-127) First the oligonucleotide (95) which contains in position 36 the codon for lie in place of Met, and the oligonucleotide (96) in which the codon for Met in position 46 has been replaced by a codon for Leu, are synthesized.

The.plasmid (72) (Figure 10) is then opened with Pvu I and Xma Ill, and the fragment (97) is isolated.

In addition, the sequence (98) in which th® codon for Met is located in front of th® codon for th® first amino acid is synthesized.

When the fragments (16), (98), (97), (95) and (96) ar® now ligated then the plasmid pS 208 (99) is obtained. This corresponds to the plasmid (72) but contains in position 0 of the CSF I sequence the codon for Met, in position 36 a codon for lie, and in position 46 a codon for Leu.

In addition, the sequence (100) which in positions 79 and 80 codes for Leu in place of Met is synthesized.

When the plasmid (76) (Figure 10) is opened with Bind III and Nhe I, and the large fragment (101) is isolated and ligated with the synthetic sequence (100), then the plasmid pS 209 (102) which corresponds to the plasmid (76) apart from replacement of the two codons in positions 79 a^d 80 in the CSF III sequence is obtained..

The plasmid (93) (Figure Ila) is now partially cut with Pvu I and with Sal I, and the large fragment (103) is isolated. The plasmid (99) is likewise partially opened with Pvu I and with Fst I, and the small fragment (104), which contains the modified CSF I sequence is isolated. In addition, the plasmid (102) is opened with Bind III and Sal I, and the small fragment (105), which'comprises the modified CSF III sequence is isolated.

The fragments (103), (104), (70) and (105) are now ligated, there being obtained the plasmid pS 210 (106) which corresponds to the plasmid (93) (Figure Ila) but codes for a CSF derivative which has Met In position 0 and in which, on the other hand, the four Met residues have been replaced by other amino acids.

When E. coli Is transformed with the plasmid (106) then, after Induction, a fusion protein is obtained which can be cleaved with cyanogen halide resulting in a CSF derivative which contains lie in position 36 and Leu in positions 46, 79 and 80.

Example 14 CSF(1-35)Ile(37-45>Leu(47-78)Leu(81-127) When the process Is carried out as in Example 13, but the synthetic sequence (107) is used in place of the synthetic sequence (100), then a deletion product which has lie in position 36 and Leu in position 46, .and in which the amino acid Leu Is present in place ox amino acids 79 and 80, is obtained. 2B Example 15 CSF(1-35) lie(37-45)Leu(47-78)-(81-127) When the process is carried out as in Example 13 but th® synthetic sequence (108) is used in place of the syn5 thetic sequence (100), then a deletion product which has lie in position 36 and Leu in position 46, and in which the amino acids in positions 79 and 80 have been deleted, in obtained.

AP] ΊΧ Synthesis block I (CSF I) (69) 1 • • ef (A w !G ATC SAC SAC CC Rg» ΛΙβ^,ΛΙ CCS SCCjCGA TOG CCS TCT CCS G C TAG CTG CTG G( O i^S ifcljtol Λ ru Uuru SSC css sct agc|ggc ! AGA see 1 $ lie Asp Asp Pi ms Ala Pro Al* Arg Ser Pre Ser Pre τ L, (1) i?’®0 * -x _ X a X& h Te • «1 δ TGG • • * TCG ACC CAG CCi GAA CAC GT T AAC GCG 1Φ fp-i CAG GA^ GC6 AGC TGG GTC GGi i*i it f* ty Λνυ g^gwiiw I vAil GTG CA A TTG CSC TAS GTC rtip R*j U£ X rtf)» yrj?y V-f \fe# t*> to ®Λ «) OxL1^ Thf* f τ^4)«ΪΙί)Μ4) &, A * to it. Jjw Gia Bis V* 1 Asn Al* lie Glu Al* ,* _ «f- 6' UO) (15) (20) wo * * * • GGT GTG CTG AAC CTG M st cgc|ga C ACS GCC GCG GAA GCA GAC GAC TTG GAC TC :a gcg ct G TGCjCGG CGC CTT Arg lea lea Asn Sea 3s sr Arg As p Thr Al* Ala Gia(25) Tk (50)'· (55) Tk 149 a> - TL n * • ' * <^b) V e • • AAC GAA ACC GTT GAA |GTG ATA TCT GAG A TG TTC GAC CTG CA TTG CTT TGG CAA CTT CAC TAT|AGA CSC S AC AAG CTG G (P at I Asa Gia Thr Val Gia Val lie'Ser Gia 1 »% Phe Asp (lea) tF ... ii40i' (<5> ——T« ί κ?θ) 1¾ W Synthesis bloekll (CSF Hi (70) 150 (psi AC -3Can.n)Gl‘ • • • COG ACA TGT stc sag acgJcg PT CTC GAG CTC TAC «*t (niW SOC Φβ1? ixi %au W Mh Ul 4f> AC A GAG GTC TGC GC :a gag! [CTO GAG ATG Ρ*·Α m> 0) V «<Εώ»ι Cys Leu Gia Thr Ar 1 g Leu Gin Leu A/ A (55) (60) FT 5 U. *» 214 , its (?» Av 0.

H • • UbX φ I M JR r ί ι GGCSCTT CGT rtQ-l φβι«ι Ql-SQ, suO eeil ¢#) rife W» gfe «’ «m (φΐφφ raw <£> Λ CCG GAA GCA jCCA AGA GAC Lys tTS-n W SAI Gly Leu Arg Gly Ser Xen 1? 4 O§ | (70 > TGG "TO Ff· T 2¾ ff»? Μ Synthesis block III (CSF III) (71) 215 III c «dU. '** ilf* &&& fweffsrn &P>f* ΑΑΙ* Ww W?U .*UxV Amm ATG ATG GCT AGC CAC TAG AAA (Bind III)A TTC CCG GGG GAG TGG TAC TAC CGA TCG GTG ATG TTT (leu)lys Gly Pro Leu Thr Met Set Ala Ses LS lye ill Bin -Wh· ’2) 75) LC ’ • • » Ij) * «1 ft UM? Λ & ,Λ| MAM TGC CCG CCG ACT CCG GAG ACG jjl A w X TGC aog| ,>RSj Λ1 rf. 'ACG SAG GTC en ofe) Λ v'l'U ACG GGC GGC TGA GGC CTC TGC AGA GGT TGC GTC Gin «is Cys w?1*· «$ Ο» β W pro t" 'S’*1 Glu Φ*5»ι we Ser Cys Ala «*») ee A Gin (90) —i ΠΓ sf - (95' ) — >1 300 .7ϊίι *n .- « • • Oi " uteri 4 • ATC # IBfl J Λ» tft |acc |»ΛΑ X Λ v fii A ·« O.&1& AAA ftf f AAC CTG AAG fe Ai ft WAV φφφ TAG TAG TGG aagI fc*ie*igp tb A AGA AAA φφβ «fe Wi SR! gSl yly ϋ * A ® A y&S'bS'b TH <<* TH * kb 1Dl*4 i® Α ώώ’’*’ -«4«S« "*·» Ser Phe Lys Glu iMjS^O Leu «if^S li. O »1 ?lx© 100) ·> .«ti- — 105) 1 hw) it! ill. k ------ SSt ei _fc-. _______ • • jisi «, * • J CTG CTT GTT ΙΦ1 CCG TT< ji^ip ΦΠΦ jJ> iSS W X W A TGG GAGjCCG GTC CA© GAA GAC GAA|cAA GGC AAJ S’ CTG ACA Λ riW)« Mwy CTC GGC SAGjGTC CTT Leu Xeu Val Tie x»> Pro Phi e Asp Cys -· ί Glu Pro Val‘Gin Glu (115) (120) _ Kt ί ;i25) ,tSl ρ lH oi •AttKKMe391 hi k Jll I Mik Ce' T «fe Γ Φ(Ζ& TAG TCG ACT GCA GCC 1 ΡΦ Μ A ATC <£7fe AGC TGA COT SGG «·>* «ϊ·! lit^ fe it? Stp ( 3d&

Claims (15)

Patent Claims 1. O exempli f ied. ,- 1 o. Human granulocyte macrophage colony-stimulating factor proteins according to claim 1, substantially as hereinbefore described and exemplified.

1. Human granulocyte macrophage colony-stimulating factor proteins (GM-CSF) of the formula Pro- (As )„»CSF( 12-126) -Z

2. - A process for the preparation of CSF as claimed in claim 1, which comprises incorporation of a gen®

3. The process as claimed in claim 2, wherein the CSF is expressed in the form of a fusion protein which 15 is then cleaved enzymatically or chemically.

4. The process as claimed in claim 3, wherein the fusion protein contains N-terminal adjacent to the CSF the amino acid sequence (Glu) ra -(Asp) n -Pro 2o in which m is zero or 1, and a is 1, 2 or 3, and is proteolytically cleaved.

5. A bacterial expression vector containing at least one gene coding for CSF as claimed in claim 1. 5 in which (As) x denotes all or some of th® first 11 amino acids of the natural GM-CSF sequence, and Z denotes Glu or Asp, hereinafter referred to aa CSF.

6. A bacterial sell, especially 2. soli, containing a 25 vector as claimed in claim S®

7. A medicament containing or composed of CSF as claimed in claim 1.

8. CSF as claimed in claim 1 for use in medical treatment.

9. The use of CSF· as claimed in claim 1 for the preparation of medicaments.

10. Coding for CSF in a bacterial expression vector, transformation of bacteria, especially E. coli, therewith, and bringing about expression therein.

11. «, A process according to claim 2 for the preparation of CSF, substantially as hereinbefore described and

12. CSF whenever prepared by a process claimed in any one of claims 2 - 4 or claim 11 .

13. A bacterial expression vector according to claim 5 ( substantially as hereinbefore described and exemplified. 15

14. A bacterial cell according to claim 6, substantially as hereinbefore described and exemplified.

15. » A medicament according to claim 7, substantially as