DK170346B1 - Granulocyte Macrophage Colony Stimulating Factor (CSF) Proteins, Method for Preparation thereof, Expression Vector and E.coli Cell for Use in This Method and Using These Proteins - 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

in DK 170346 B1

Human granulocyte macrophage "Colony-Stimulating," - factor (GM-CSF), hereinafter referred to as "CSF", is a glycoprotein having a molecular weight of about 23,000 Daltons. The cDNA sequence and expression of the glycoprotein in mammalian cells is already known (GG Wong et al., Science 228 (1985), 810-815, D.

Metcalf, Science 229 (1985), 16-22).

The invention relates to gramilocyte macrophage "Colony Stimulation" factor (CSF) proteins, which are available by known variations of the DNA sequences. Thus, for example, in the construction of vectors for fusion proteins, cleavage sites can be incorporated which, after cleavage of the CSF protein, show C-terminal and / or N-terminal variations in the amino acid sequence. The invention also relates to a method for producing CSF proteins according to the invention. In addition, the invention relates to the use of CSF proteins of the invention for the manufacture of drugs and drugs containing or consisting of CSF proteins of the invention, in particular drugs for stimulating the proliferation of haematopoietic cells and for promoting ganulocyte-20 and macrophage formation. .

The invention further relates to expression vectors for use in bacteria, especially in E. coli, which contain a DNA operatively linked to CSF or CSF fusion protein.

Further aspects of the invention and preferred embodiments are therefore elucidated in the following or defined in the claims.

In order to illustrate the invention, figures 1-15 of the drawing are also used, in which in each case, mostly in the form of a flow chart, the methods of the examples with the same number are illustrated. These figures are not on the correct scale, especially in the polylinker range the scale is "extended".

Thus, in FIG. 1 and sequences 35 1a and 1b thereof, the preparation of the vector pW 225, which serves to directly express (Met-) CSF. The following 2 DK 170346 B1

ISLAND

Figures relate to vectors leading to expression of fusion proteins in whose N-terminal a "ballast" protein is placed in front of the CSF amino acid sequence, and this protein is derived from a human sequence interleukin-2,5 subunit below. designated "IL-2" or "/ ^ EL-2": * In FIG. 2 and sequences 2a and 2b thereof, the preparation of the vector pW 216 encoding a fusion protein is shown from which, upon acid cleavage, a CSF derivative, which is N-terminal, is extended by the 10 amino acid proline.

In FIG. 3 shows the synthesis of the vector pW 240, which encodes a fusion protein which, after acid cleavage, leads to a CSF derivative which carries the proline instead of the solid amino acid (alanine).

FIG. 4 relates to the preparation of the vector pW 241, which encodes a fusion protein which, after acid cleavage, leads to a CSF derivative in which the first amino acid (alanine) is missing.

In FIG. 5 shows the preparation of the vector pW 242, 20 which encodes a fusion protein which, after acid cleavage, leads to a CSF derivative in which the first 5 amino acids are removed.

FIG. 6 relates to the preparation of the vector pW 243 encoding a fusion protein which, after acid cleavage, 25 leads to a CSF derivative in which the first 7 amino acids are missing.

In FIG. 7, the synthesis of the vector pW 244 encoding a fusion protein is shown by which, after acid cleavage, a CSF derivative is obtained in which the first 11 amino acids are eliminated.

In FIG. 8 and continuation 8a thereof, the synthesis of the vector pW 246 is shown. This encodes a fusion protein in which, following the IL-2 sub sequence, two variants are designated as "CSF" designated sequences. After acid cleavage, a CSF derivative is obtained in which proline is the N-terminal front of the first amino acid and in which it is.

last amino acid has been replaced with aspartic acid.

In FIG. 9, the synthesis of the vector pW 247 / which encodes a fusion protein is shown in which, following the IL-2 sub-sequence, three CSP 'sequences follow. After acid cleavage, it is obtained in FIG. 8 characterized CSF derivative.

In FIG. 10 and the continuation; FIG. 10a, the preparation of the hybrid plasmids pS 200-204, which has synthetic CSF DNA sub-sequences, is shown, the plasmid pS 200 containing "Synthesis block I" of Annex I, the plasmid pS 201 10 containing "Synthetic block II" of Annex II, the plasmid pS 202 contains "Synthesis block III" according to Appendix III, the plasmid pS 203 contains the entire synthetic gene and pS 204 is an expression plasmid which also contains the entire synthetic CSF DNA sequence. After expression and acid cleavage, the same CSF derivative as described in FIG. 2nd

In FIG. 11 and the continuation, FIG. 11a, there is shown the synthesis of the expression plasmid pS 207, which encodes a fusion protein which, after cleavage with N-bromosuccinimide, yields a CSF derivative in which Trp at positions 13 and 20122 is replaced each time by His.

In FIG. 12, a synthetic DNA part sequence is shown which allows the preparation of CSF derivative, in which Ile at position 100 is replaced by Thr.

In FIG. 13 and the continuation, FIG. 13a, there is shown the synthesis of the expression plasmid pS 210, which encodes a fusion protein which, upon cyanobromide cleavage, yields a CSF derivative in which all methionine groups are replaced by neutral amino acids, namely at position 36 of Ile and at positions 46 , 79 and 80 by Leu.

In FIG. 14 shows a synthetic DNA sequence which according to the synthetic scheme of FIG. 13 allows the preparation of a CSF derivative in which Met at position 36 is replaced by Ile and at position 46 by Leu, and instead of amino acids 79 and 80 there is a single Leu group.

35 In FIG. 15 finally shows a synthetic DNA whose use in the synthesis scheme of FIG. 13 enables the position of a CSF derivative in which Mét at position 36 is replaced by Ile and position 46 by Leu and wherein the two amino acids at positions 79 and 80 are removed. ♦

It has been found that the "open reading lattice" 5 of a DNA encoding 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, as in substantially equal to the first hundred amino acids, is particularly well suited for the production of fusion proteins.

Thus, as a major product, one obtains a fusion protein, which completely or to a large extent consists of eukaryotic protein sequences. Surprisingly, this protein is obviously not recognized as foreign protein by the host proteases and is not immediately degraded again.

It is a further advantage that the fusion proteins of the invention are heavily soluble to insoluble and hence "simply separable from the soluble proteins, conveniently by centrifugation. because the IL-2 moiety is a biologically active molecule, it does not depend on the exact structure of the IL-2 moiety as well. For example, it is possible, for example, to carry out variations at the N-terminal which allow a cleavage of the fusion protein if the desired protein is arranged thereto N-terminally, Conversely, C-terminally, variations can be made to enable or facilitate the cleavage of the desired protein.

The natural DNA sequence encoding human IL-2 is known from EP-A No. 0.091.539. The literature cited refers to mouse and rat IL-2. These mammalian DNAs may be considered for the synthesis of the proteins of the invention. Conveniently, however, one assumes a synthetic DNA, particularly advantageously from a human IL-2 DNA, which is disclosed in German publication specification no.

DK 170346 Bl 5 3.419.955 or in EP-A 0.163.249. This synthetic DNA not only has the advantage of being matched in the KODON range to the conditions of the most frequently used host, E. coli, but it also contains a number of cleavage sites 5 for restriction endonucleases at the beginning or in the range for the 100th triplet which can be used according to the invention. However, this does not exclude the possibility that variations in the DNA can be made in the intermediate region, making use of the additional 1 ° gap sites.

Thus, if you use the nucleases Ban II, Sac I or Sst I, you get an IL-2 sub-sequence which encodes approx. 95 amino acids. This length is usually sufficient to obtain an insoluble fusion protein. For example, if, for example, a desired hydrophilic CSF derivative is not sufficiently soluble, but one does not - to produce as little "ballast" as possible - but makes use of the cleft sites adjacent to the C-terminal, one can thus corresponding adapters or linkers extend the DNA sequence at the N- and / or C-terminal end, thus "cleaving the" ballast "portion on target". Of course, one can also use the DNA sequence - more or less - to the end, thus forming - possibly modified - biologically active IL-2 as a "by-product".

The CSF molecules of the invention are characterized in that they have the general formula Pro- (As) X-CSF (12-126) -Z, wherein (As) x, in whole or in part, means the first 11 amino acids of the natural CSF- sequence and Z means Glu or Asp.

The CSF proteins at issue are essentially known to contain the amino acid proline at the N-terminal.

In contrast to naturally occurring human GM-CSF, these CSF proteins do not occur in the human body.

In this connection, it should be emphasized that human GM-CSF 35 does not naturally act antigenically in the human body, as otherwise an autoimmune reaction would occur in the body, which is not the case in healthy humans either. Based on the article by Benjamin et al. (1984), it is surprising that the CSF proteins in question, which do not occur in nature, do not appear antigenic, as Benjamin et al. already 5 have shown that antibodies are formed against highly conserved proteins which differ only from naturally occurring proteins with respect to a single amino acid. It is precisely because of the lack of antigenic potential of the CSF proteins in question that it is at all possible to use these 10 as pharmaceuticals for humans. In this regard, they are even superior to the naturally occurring human GM-CSF, as they are more stable to proteases than human GM-CSF.

The method of the invention is characterized by incorporating a gene encoding CSF into a bacterial expression vector, thereby transforming bacteria, especially E. coli, into the gene for expression therein.

The cleavage of the fusion protein can occur in a manner known per se, chemically or enzymatically. The choice of the appropriate method is primarily directed to the amino acid sequence of the desired protein. Thus, in the bridging element Y at the carboxy terminal tryptophan or methionine or Y Trp or Met, a chemical cleavage with N-bromosuccinimide or halogencyan can occur if CSF derivatives containing each of these 25 amino acids are synthesized each time.

CSF and its derivatives, which in their amino acid sequence contain Asp-Pro and are sufficiently acid stable, can be proteolytically cleaved in a manner known per se, as already shown above. This gives proteins containing N-terminal proline and C-terminal aspartic acid. Thus, modified proteins can also be synthesized in this way.

The Asp-Pro bond can be formed even more acid labile when this bridge element is (Asp) n ~ Pro or 35 Glu- (Asp) n-Pro, with n being 1-3.

7 DK 170346 B1

Examples of enzymatic cleavages are also known in that modified enzymes with improved specificity can also be used (cf. C.S. Craik et al., Science 228 (1985) 291-297).

The fusion protein is recovered by expression in a bacterial expression system in a manner known per se.

To this end all known host vector systems such as bacteria of the species Streptomyces, B. subtilis, Salmonella typhimurium or Serratia marcescens, especially E. coli, are suitable.

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

In this connection, the promoter and operator are conveniently selected from trp, lac, tac, PL or PR 15 from the phage hsp, omp or a synthetic promoter, as they are, for example, proposed in German publication no. 3,430,683 or in EP-A no. 0.173.149. Advantageously, the tac promoter-operator sequence is commercially available (e.g., expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, p. 63).

In the expression of the fusion protein of the invention, it may be appropriate to modify single triplets in the first amino acids after the ATG start-25 codon to prevent any base pair formation on the mRNA plane. Such changes as removing or adding single amino acids are well known to those skilled in the art, and the invention also relates to these changes.

CSF proteins are preferred which, in addition to the N-terminally added proline, have the entire CSF amino acid sequence. However, it has surprisingly been found that the variations of the CSF molecule obtained by elimination of the first eleven amino acids are also biologically active.

Also advantageous are variants of the invention which first lead to fusion proteins containing the CSF sequence more than once, advantageously two or three times. In these fusion proteins, the ballast moiety is naturally diminished and the yield of the desired protein then increased.

At American Type Culture Cellection, the plasmid pHG 23 is deposited under the number ATCC 39900 in E. coli, and this plasmid 5 is obtained by incorporation of the CSP cDNA sequence into the Pst I cleavage site * "of pBR 322. The DNA sequence corresponds to the The variant described in Fig. 3 (B) by Wong et al., for incorporation, uses the Pst I cleavage site near the 5 'end on one side and a Pst I site introduced by GC "Tailing" at 10 3 'end (EP-A 0.183.350).

Example 1 Pro ° -CSF

(5 The vector pUC 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 fragment (2) (Example 1? Figure 1) Competent cells of E. coli JM 103 are transformed with the ligation mixture, and 20 of the desired clones containing the plasmid pW 212 (18) are selected.

From plasmid DNA, fragment (19) is cleaved with Pvu I and Pst I, and this fragment contains the CSF sequence.

By inserting the lac repressor (PJ Parabaugh, 25 Nature 274 (1978) 765-769) into plasmid pKK 177-3 with the pUC 8 polylinker (Amann et al., Gene 25 (1983) 167? EP-A No. 0.133 .282) gives the plasmid pJF118 (20) (Fig. 1; cf. German Patent Application No. P 3,526,995.2, Example 6, Fig. 6). This is opened at the singular restriction site 30 of Ava I and is reduced in a manner known per se by exonuclease treatment to approx. 1000 bp. After ligation, the plasmid pEW 1000 (21) is obtained, whereby the lac reporter gene is completely obtained, which, however, due to the decrease, has a significantly larger number of copies than the starting plasmid.

Instead of the plasmid pKK177-3, the above-mentioned commercially available plasmid pKK223-3 can also be used, incorporate the lac repressor and shorten the produced product by analogy.

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

Plasmid pl59 / 6 (23), prepared according to German Publication No. 3,419,995 (EP-A No. 0.163,249), Example 4 (Fig. 5), is opened with the restriction enzymes 10 Eco RI and Sal I, and the small fragment (24) is isolated and this fragment contains the IL-2 sequence.

By ligating the fragments (22) and (24), the hybrid plasmid pEW 1001 (25) is obtained.

The plasmid (25), on the one hand, is opened with Eco RI 15 and Pvu I to give fragment (26), which contains the major part of the IL-2 sequence. This part sequence is denoted in the figures by "/ \ IL2".

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

By ligating the fragments (19), (26) and (27), transformation of competent E. coli 294 cells and selection yield clones containing the plasmid pW 216 (28). Plasmid DNA is characterized by restriction and DNA sequence analysis.

A culture of overnight E. coli cells containing the plasmid (28) is diluted with LB medium (JH Miller, see above) containing 50 µg / ml ampicillin at a ratio of approx. 1: 100, and growth is monitored via OD measurement. At OD = 0.5, culture is adjusted to 30 1 mM isopropyl-3-galactopyranoside (IPTG) and the bacteria are centrifuged after 150-180 minutes. The bacteria are boiled for 5 minutes in a buffer mixture (7 molar urea, 0.1% SDS, 0.1 molar sodium phosphate, pH = 7.0) and samples are applied to an SDS gel electrophoresis plate.

After electrophoresis, a protein band is obtained from bacteria containing the plasmid (28) and this band corresponds to the size of the expected fusion protein. After picking up the bacteria (French Press; Dyno-Miihle) and centrifuging, the fusion protein is in the precipitate / so that already in the above liquid considerable amounts of the other proteins can be separated. After isolation of the fusion protein, the expected CSF derivative containing additional N-terminal proline / by acid cleavage is released. This shows activity in the biological test.

The indicated induction conditions apply to shaking cultures; For larger fermentations, correspondingly altered OD values and possibly slightly different IPTG concentrations are appropriate.

Example 2 Pro1-CSF (2-127)

By ligating the fragments (2) (Fig. 1) and (16) (Fig. 2) with the synthetic DNA sequence (29), the hybrid plasmid (30) is obtained which corresponds equally with the exception of the synthetic DNA sequence. the plasmid (18).

The plasmid (30) is cleaved with Pvu I and Pst I

however, the fragment (31) containing the CSF DNA sequence in which the codon for the first amino acid is replaced by a codon for protein. By ligating the fragment (31) to fragments (26) and (27), the hybrid plasmid 25 pW 240 (32) is obtained. The expression in E. coli carried out according to Example 1 gives a CSF derivative in which the first amino acid is replaced by proline. This derivative also has biological activity.

Example 3 CSF (2-127)

A plasmid containing the CSF DNA sequence with a Pst-I restriction site at the 3 'end thereof, for example plasmid pHG 23 (ATCC 39900), is digested with Sfa NI,

35 and the linearized plasmid (34) is partially fulfilled by Klenow polymerase and GTP. The protruding nucleotide A

o 11 DK 170346 B1

From the plastic cut (43), the fragment (44) is isolated with Pvu I and Pst I, and this fragment contains the DNA sequence of the CSF derivative. This fragment (44) is ligated to fragments (26) and (27), leading to the formation 5 of the hybrid plasmid pW 242 (45).

The expression of Example 1 leads to the formation of a fusion protein from which, after acid cleavage, a CSF derivative is obtained in which the first five amino acids are missing. This product is also biologically active.

10 is cleaved with S1 nuclease and then the fragment (35) is cleaved with Pst I.

By ligating the fragment (35) with the synthetic DNA sequence (36) and the fragment (16) (Fig. 2), the plasmid (37) analogous to the plasmid (18) is obtained.

From the plasmid (37), the fragment (38) is cleaved by Pvu I and Pst I. This fragment is ligated with the fragments (26) and (27) to give the plasmid pW 241 (39).

By expression of Example 1, fusion protein is obtained, which after acid cleavage gives a CSF derivative in which the first amino acid is missing. This derivative is biologically active.

Example 4 CSF (6-127).

The plasmid (33) (or a similar plasmid containing the CSF - * DNA sequence) is first cleaved completely with Pst I and then partially with Bst NI and the fragment (40) isolated.

The synthetic DNA sequences (41) and (36) (Fig. 4) are first ligated to the sequence (42) and then ligated to the fragment (16) (Fig. 2) and the fragment (40) to obtain the plasmid. pW 212 (43).

35 0 12 DK 170346 B1

Example 5 CSF (8-127).

If one first compares the synthetic DNA sequence (36) (Fig. 4) with the synthetic DNA sequence (46) and then the DNA fragment (47) thus obtained with the fragments (40) and (16) is thus obtained. the hybrid plasmid (48).

From this, the Pvu I and Pst II fragment (49), which contains the DNA sequence of the CSF derivative, is cleaved.

Ligation of fragments (49, (26) and (27) yields the hybrid 10 plasmid pW 243 (50) which corresponds to the plasmid (45) equally except for the abbreviated DNA sequence of the CSF derivative.

The expression of Example 1 leads to a fusion protein which, after acid cleavage, yields a CSF derivative in which the first seven amino acids are lacking. This derivative is also biologically active.

Example 6 CSF (12-127).

If one synthesizes the synthetic DNA sequence (51) with the fragments (33) and (16), the hybrid plasmid (52) is thus obtained. If this is cleaved by the Pvu I and Pst I sequence (53) containing the DNA sequence of the CSF derivative and ligated this fragment (53) with the fragments (26) 25 and (27), the hybrid plasmid pW is thus obtained 244 (54) corresponding to the plasmid (45) equally except for the abbreviated CSF sequence.

The expression of Example 1 leads to a fusion protein which, after acid cleavage, yields a CSF derivative in which amino acids 1-11 are removed. This abbreviated molecule is also biologically active.

Example 7 -

Pro ° -CSF (1-126) -Asp.

35 The DNA sequence (19) (Fig. 2) is partially cleaved with Bst NI and the fragment (55) containing the major portion of the CSF sequence is isolated.

Upon cleavage of the plasmid (33) (Fig. 4) (or a similar plasmid containing the CSF DNA sequence) with first Pst I and then partially with Bst NI, the DNA-5 sequence (56) comprising the most of the CSF sequence.

The DNA sequence (57), which completes the sequence (56), is synthesized into a DNA sequence encoding CSF derivative in which the C-terminal glutamic acid is replaced by 1o aspartic acid.

The vector pUC 13 is opened with Pst I and Sma I, isolating the large fragment (58). Thus, if this residual plasmid (58) is compared with fragments (56) and (57), the hybrid plasmid pW 245 (59) is obtained with the C-terminally modified sequence.

From the plasmid (59) the fragment (60) containing the modified CSF DNA sequence is cleaved by Sfa NI and Pst I. This fragment (60) is ligated to the synthetic DNA sequence (61) and fragment 20 (55) to give the DNA sequence (62). This is ligated to the DNA fragments (26) and (27) (Fig. 2) to give the hybrid plasmid pw 246 (63). This plasmid is reproduced twice in FIG. 8a, taking the amino acid sequence of the coding fusion protein from the lower representation.

By the expression of Example 1, a fusion protein is obtained, of which, after acid cleavage, a CSF derivative is obtained which is N-terminally extended with proline and in which the last amino acid is also replaced by aspartic acid. This derivative is biologically active.

30

Example 8 Pro ° -CSF (1-126) -Asp

The hybrid plasmid (63) (Fig. 8) is cleaved with Eco RI and Pst I and isolated the fragment which, in association with the IL-2 sub sequence, contains the two modified CSF sequences. This sequence (64) is cleaved DK 170346 B1 14

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partially with Rsa I, and the two fragments (65) and (66) are isolated. By ligating the DNA sequences (27), (65), (67), (61) and (60), the hybrid plasmid pW 247 (68) is obtained in which the ligated sequences are arranged in the order mentioned.

The expression of Example 1 gives a fusion protein from which, after acid cleavage, the same CSF derivative as of Example 7 is obtained.

Example 9 Synthetic gene (for Pro ° -CSF).

By methods known per se, for example, according to the phosphite method (German Publication No. 3,327,007, 3,328,793, 3,409,966, 3,414,831 and 3,419,995), the three "synthesis blocks" I (CSF-I) are synthesized in Figures .jg designated as (69), II (CAF-II) j in Figures as (70), and III (CSF-III); in the figures as (71). In the nucleotide sequence of these synthesis blocks, the synthesized oligonucleotides Ia-Im, Ila-IIf and Illa-IIIl are plotted (appendix).

With the choice of the nucleotides for the synthetic gene, not only are singular cleavage sites determined at the junction sites of the three synthesis blocks, but within the gene fragments also a number of singular restriction sites. These are listed in the following tables. These singular restriction sites may, in a manner known per se, serve to replace, supplement or remove amino acid codons.

30 35 0 15 DK 170346 B1

Site block I (CSF I) - Cleavage by nucleotide no.

Enzyme Recognition Sequence (coding strand)

When I GG + CGCC 1 5 Hpa II C + CGG 4 'Hae II GGCGC4C 4

Nae I GCC4GGC 5

Pvu I CGAT + CG 13

Will I G + TCGAC 24 10 Acc I GT4CGAC 25

Hine II GTC4GAC 26

Hpa 1 / GTT4AAC 48

Hine II

Hha I GCG40 66 15 Hinf I G + AGTC \ 88

Nru I TCG + CGA 89

Xma III ClGGCCG 95

Sac II CCGC4CG 101

Eco RV GAT4ATC 128 20

Synthesis Block II (CSF-II) Cleavage by Nucleotide no.

Enzyme Recognition Sequence (coding strand)

Afl III A4CATGT 157

Mlu I A4CGCGT 169 30 Xho I ClTCGAC 175

Take I T + CGA 176

Hga I GACGC (5/10) 177

Ava I C + TCGAG 177

Alu I AG + CT 180 35 Sac 1 / GAGCT + C 182

Hgi AI

Stu 1 / AGG + CCT 194

Hae I

0 16 DK 170346 B1

Synthetic Block III (CSF-III)

Cleavage by Nucleotide No. Enzyme Recognition Sequence (coding strand) 5 Breed II C + TTAAG 217 - Hae III GG + CC 224

Monkey I GGGCC + C 227

Mnl I CCTC (7/7) 238

Nhe I G * CTAGC 241 10 Mae I C + TAG 242

Aha II GA + CGTC 280

Aat II GACGT4C 283

Sci NI G4CGC 287

Mst I TCG + GCA 288 15 Sau 3AI / IGATC 296

Mb o I

Dpn I GA + TC 298

Asu II TT + CGAA 308

Aha III TTT + AAA 318 20 Ava II G4GTCC 382

Eco RI I + CCAGG 384

Bst NI / CC + AGG 380

Ser PI

The three synthesis blocks are first cloned individually, increased in E. coli and isolated again:

The synthesis block CSF-I (69) is incorporated into the pUC 12 derivative (16) to give the plasmid pS 200 (72).

pUC 12 is opened with the restriction enzymes Pst I and 30 Hind III and ligated with the linearized plasmid (73) from the synthesis block CSF-II (70) to give the plasmid pS 201 (74).

pUC 13 is opened with Hind III and Sma I, and the linearized plasmid (75) is ligated with CSF-III to give the plasmid pS 202 (76).

0 17 DK 170346 B1

The reinsulated synthesis blocks (69) / (70) and (71) are then ligated into the Eco-RI and Sma II linearized vector pUC 12 (77) to obtain plasmid pS 203 (78).

This hybrid plasmid is increased - such as the plasmids with the single synthesis blocks - in E. coli 79/02, and the synthetic gene is characterized by restriction and sequence analysis.

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

The expression plasmid (21) is opened with Eco RI and

Child HI, and the large fragment (80) is isolated. This fragment (80) is then ligated to the fragment (26) containing the IL-2 sub sequence and the synthetic gene (79). Thereby the plasmid pS 204 (81), which encodes a fusion protein, is obtained, in which, on the IL-2 sub sequence, the bridge element which allows the acid cleavage is followed first and then the amino acid sequence of CSF. Accordingly, by acid cleavage a CSF derivative is obtained which is N-terminally extended by proline.

Example 10 CSF (1-12) His (14-121) His (123-127)

Substituting in synthesis block I oligonucleotides 1a and 1b as well as Ic and 1d with the synthetic sequences (82) and (83), a modified synthesis block I is thus obtained as co ..

25 for a CSF-I analog in which Trp is in front of the first amino acid (Ala) and at position 13, Trp is replaced by His.

The plasmid (72) (Fig. 10) is opened with Eco RI and Hpa I, and the large fragment (84) is isolated. This is then ligated to the synthetic fragments (82) and (83) to give the plasmid pS 205 (85) encoding this modified CSF I- (CSF-11).

The plasmid (76) (Fig. 10) is opened with Hind III and Sal I and the small (86) and large (87) fragment isolated. The small fragment (86) is cleaved with Taq I} and

OC

the fragment (88) is isolated.

The large fragment (87) is then ligated with (88) and the synthetic fragment (89) in which the codon for Trp at position 122 is replaced with His, yielding the plasmid pS 206 (90), which encoding the modified CSF III (CSF-III ') · This plasmid is transformed, amplified and re-isolated after E. coli, cleaved with Hind III and Sal I,' and the small fragment (91) encoding CSF III ', isolated.

The plasmid (85) is partially cleaved with Pvu I and with Pst I, and the small fragment (92) encoding CSF-I 'is isolated.

If fragments (22), (26), (92), (70) and (91) are then ligated, the plasmid pS 207 (93) is thus obtained. This encodes a fusion protein in which on the IL-2 partition sequence follows a bridge element which immediately in front of the first amino acid of CSF (Ala) contains Trp. Since Trp in the CSF-15 molecule at positions 13 and 122 is replaced by His, the fusion protein can be cleaved with N-bromo-succinimide. This gives the CSF derivative, in which tryptophan in both positions is replaced by histidine.

Example 11 CSF (1-99) Thr (101-127)

Replacing by the synthesis of the synthesis block III the oligonucleotides Ille and Ulf with the synthetic sequence (94); and if otherwise proceeded according to Example 9, there is thus obtained a CSF derivative in which ILe at position 100 is replaced by Thr.

Example 12 CSF (1-35) Ile (37-45) Leu (47-78) Leu-Leu (81-127) The oligonucleotide (95) containing codon for Ile instead of Met is first synthesized. and the oligonucleotide (96), in which the codon for Met is replaced by Leu. .

Then, plasmid (72) (Fig. 10) is opened with Pvu I 35 and Xma III, and the fragment (97) is isolated. Further, the sequence (98) is synthesized in which prior codon for the first amino acid is codon for Met.

If fragments (16), (98), (97), (95) and (96) are then ligated, the plasmid pS 208 (99) is thus obtained. This is similar to the plasmid (72) but contains in the CSF-I sequence at position 0 codon for Met, at position 36 a codon for Ile and at position 46 a codon for Leu.

Furthermore, the sequence (100), which in positions 79 and 80 encodes Leu instead of Met, is synthesized.

10 Opens the plasmid (76) (Fig. 10) with Hind III

and Nhe I, isolating the large fragment (101) and ligating it to the synthetic sequence (100) thus obtains the plasmid pS 209 (102) corresponding to the plasmid (76) except for the exchange of the two codons at position 79 and 80 in the CSF-III-15 sequence.

The plasmid (93) (Fig. 11a) is then partially cleaved with Pvu I and Sal I and the large fragment (103) is isolated. Plasmid (99) is also partially opened with Pvu I and with Pst I, isolating the small fragment (104) 20 containing the modified CSF-I sequence. In addition, the plasmid (102) is opened with Hind III and Sal I and isolated the small fragment (105) comprising the modified CSF-III sequence.

Then, the fragments (103), (104), Ϊ70) 25 and (105) are ligated to give the plasmid pS 210 (106), which corresponds to the plasmid (93) (Fig. 11a), but encodes a CSF derivative , in which Met is in position 0, and on the other hand, the four Met groups are replaced by other amino acids.

Thus, when transformed with the plasmid (106) E. coli, 30, upon induction, a fusion protein cleavable with cyanohalide is obtained, yielding a CSF derivative containing at position 36 Ile and at positions 46, 79 and 80 Leu. .

35 DK 170346 B1 20 o

Example 13 CSF (1-35) Ile (37-45) Leu (47-78) Leu (81-127) Proceed according to Example 12, but instead of the synthetic sequence (100), the synthetic sequence 5 (107) is used. ), there is thus obtained a deletion product in which at position 36 stands Ile and at position 46 Leu and in which amino acids Leu are substituted for amino acids79 and 80.

Example 14 CSF (1-35) Ile (37-45) Leu (47-78) - (81-127) Proceed according to Example 12, but instead of the synthetic sequence (100) insert the synthetic sequence (108 ) thus obtains a deletion product wherein 15 at position 36 stands for Ile and at position 46 Leu, and the amino acids at positions 79 and 80 are removed.

20 25 "30 35 21 DK 170346 B1 0

Appendix

Synthesis Block I (CSF-I) (69) 5 - Ici ----- Ic 1 · ·

• AAT TCG ATC GAC GAC CCG GCG CCG GCcJcGA TCG CCG TCT CCG

GC TAG CTG CTG GGC CGC GGC CGG GCT AGcJgGC AGA GGC

(Eco Ri) Ile Asp Asp Pro Ala Pro Ala Arg Ser Pro Ser Pro

io »--xt — LU_122. ^ -1J

t 50 XC - ► - 2nd - -—X-3

TCG ACC CAG CCC | tGG GAA CAC GTT AAC GCG AT C CAG GaJa GCG 15 AGC TGG GTC GGG ACC CTT | GTG CAA TTG CGC TAG GTC CTT CGC

Ser Thr Gin Pro Trp Glu His Val Asn Ala Ile Gin Glu Ala τά— ^ ι - m_if

1 \ H ------

20 c · · · ·

CGG CGTCTG CTG AAC CTG AGT CGC | GAC ACG GCC GCG GAA ATG

Gcjc GCA GAC GAC TTG GAC TCA GCG CTG TGC | CGG CGC CTT TAC

Arg Arg Leu leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met li - «- £ 22) -Xf, - (30) - ^ - - (35) -1 k 25 --U_!« • · · · ·

AAC GAA ACC GTT GAAJgTGATA TCT GAG ATG TTC GAC CTG CA

TTG CTT TGG CAA CTT CAC TATjAGA CTC TAC AAG CTG G (Pst I) 30 Asn Glu Thr Val Glu Val Ile Ser Glu With Phe Asp (leu) T / (40) (45) (50)

Xk ---- ► ---- p * 35 * 22 DK 170346 B1 0

Synthesis block II (CSF-II) (70) 150 ^ τη-.

- HA- --- JlC

5 · · · · ** (Pst i) g gaa ccg aca tgt ctc cag acg | cgt ctc gag ctc tac

AC GTC CTT GGC TGT ACA GAG GTC TGC GCA GAG | CTC GAG ATG

(Gln) Glu Pro Thr Cys Leu Gin Thr Arg Leu Glu leu Tyr (50) __ (55) (60) _.

10 JLL ia 1 "pm ............... II Cl _ 200 w 214 n c --- - * - jPe-► *> AAA CAA GGCjCTT CGT GGT TCT CTG ACC A ( Hind III)

TTT GTT CCG GAA GCAlCCA AGA GAC TGG TTC GA

15 Lys Gin Gly Leu Arg Gly Ser Leu Thr (Lys)

Icl - <«> --- iZSljrf-- 20 25 30> 35 0 23 DK 170346 B1

Synthesis Block III (CSF-III) (71) 215 ΤΓ 250 _ ^ —...... ilia ----- JITc • · · · ·

5, AG CTT AAG GGG CCC CTC ACC ATG ATG GCT AGC CAc | tAC AAA

("Hinå III) A TTC CCC GGG GAG TGG TAC TAC CGA TCG GTG ATG TTT | (Leu) list Gly Pro leu Thr Met With Ala Ser His Tyr list (72) (75) (80) (85) --BTfc- - 10 ffic - - - *** · --UTe

CAG CAC TGC CCG CCG ACT CCG GAG ACg | tCT TGC GCA ACG CAG GTC GTG ACG GGC GGC TGA GGC CTC TGC AGA ACg] CGT TGC GTC

Gin His Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr Gin, 5 --- ^ £ L_sN-r (95) ► * -sr-f 300 _ jTe-7 —-; - J-ϋί-3 -; - -

ATO ATCIACC TTO GAA TO TTT AAA GAA AAC CTS AAG GAC ITT

TAG TAG TGG AA&I CTT AGA AAA TTT CTT TTG GAC TTC CTG AAA

20

Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe _-Ooo) (105) (110) j! Lf - Hi 350 391 --Ml i ------ nrk • · · · ·· ^

25 | cTG CTT GTT ATA CCG TTC GAC TGT TGG GAG | CCG GTC CAG GAA

GAC GAA | CAA TAT GGC AAG CTG ACA ACC CTC GGC CAG | GTC CTT

Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gin Glu (115) (120) (125).

Dpi - l & j -► - / II i

30 V

Mk— »

Will I Pst I TGA TA * G TCG ACT GCA GCC C ACT ATC AGC TGA CGT CGG G 35 Stp Stp (Sma I)

Ri ^

Claims (8)

24 DK 170346 B1 PATENT REQUIREMENT. 1. Granulocyte macrophage "Colony Stimulating" factor (CSF) proteins, characterized in that they have the general formula Process for producing CSF according to claim 1, characterized in that a gene encoding CSF encodes a bacterial expression vector, thereby transforming bacteria, especially E. coli, and bringing the gene into expression therein. Process according to claim 2, characterized in that CSF is expressed in the form of a fusion protein, which is then enzymatically or chemically cleaved. Method according to claim 3, characterized in that the fusion protein N-terminal adjacent to the CSF contains the amino acid sequence (Glu) m- (Asp) n-Pro, wherein m is zero or 1 and η 1, 2 or 3, and the proteo is cleaved. -lytisk. Bacterial expression vector, characterized in that it contains at least one gene encoding CSF according to claim 1. Pro- (As) χ-CSF (12-126) -Z, wherein (As) x represents, in whole or in part, the first 11 amino acids of the natural CSF sequence, and Z means Glu or Asp. A bacterial cell, especially E. coli, characterized in that it contains a vector according to claim 5. Medicament, characterized in that it contains or consists of CSF according to claim 1. Use of CSF according to claim 1 for the manufacture of drugs. 35