CA1341197C - Gm-csf protein, its derivatives, the preparation of proteins of this type, and their use - Google Patents
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Abstract
Expression of a gene coding for human granulocyte macrophage colony-stimulating factor (CSF) in bacteria results in CSF proteins which are biologically active.
Modification of the natural or of a synthetic gene structure results in biologically active derivatives with a modified amino acid sequence.
Modification of the natural or of a synthetic gene structure results in biologically active derivatives with a modified amino acid sequence.
Description
HOECHST AKTIENGESELLSCHAFT HOE 85/F 293 Dr.KL/ml GM-CSF protein, its derivatives, the preparation of proteins of this type, and their use 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 expres-sion of the glycoprotein in mammalian cells have already been disclosed (G.G. Wong et al., Science 228 (1985>, 810-815, D. Metcalf, Science 229 (1985), 16-22).
It has now been found, surprisingly, that the expres-sion of human GM-CSF protein, called "CSF" hereinafter, in bacteria results in a biologically active product.
Thus the invention relates to CSF for use in medical treatment and to the use for the preparation of medi-cements.
The invention furthermore relates to the preparation of CSF by expression in bacteria, in particular in E.
coli. In particular, it is possible to use for this purpose the published cDNA sequences which can be ob-tained in a manner known per se, preferably by synthe-sis.
The invention additionally relates to expression vectors for use in bacteria, in particular in E. coli, which contain, in a suitable arrangement ("operatively linked to">, a DNA coding for CSF or a CSF fusion pro-tein.
The invention additionally relates to biologically active derivatives of CSF which can be obtained by modifications, which are known per se, of the ONA
sequences. Thus, for example, it is possible to in-corporate cleavage sites in the construction of ~3'~~19~
vectors for fusion proteins which, after elimination of the CSF protein, have C-terminal and N-terminal modifi-cations in the amino acid sequence. Furthermore, the invention relates to the use of proteins of this type in medical treatment and to their use for the prepara-tion of medicaments, and to medicaments which contain CSF protein and its biologically active derivatives, in particular medicaments for the stimulation of prolifer-ation of hemopoietic cells and for promotion of the formation of granulocytes and macrophages.
Further aspects of the invention and its preferred em-bodiments are illustrated in detail below and are defined in the patent claims.
The 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 the same numbers. These figures are not to scale, in particular the scale has been "expanded" in the region of the polylinkers.
Thus, Figure 1 and its continuations 1a and 1b show the preparation of the vector pW 225 which is used for the direct expression of (Met-)CSF. The figures which fol-low relate to vectors which result in the expression of fusion proteins in which a "ballast" protein, which is derived from a part-sequence of human interleukin-2, hereinafter "IL-2" or "eIL-2", is located at the N-terminal end in front of the CSF amino acid sequence:
Figure 2 and its continuations 2a and 2b show the prep-aration 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 pW 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 pW 241 which codes for a fusion protein which results, after S acid cleavage, in 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 acids have been eliminated.
Figure 6 relates to the 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 in a CSF
derivative in which proline is located at the N-termi-nal end in 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' sequen-ces 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 pre-paration of the hybrid plasmids pS 200 to 204 which contain synthetic CSF DNA part-sequences, the plasmid pS 200 containing "synthesis block I", shown in Appen-dix I, plasmid pS 201 containing "synthesis block II"
shown in Appendix II, plasmid pS 202 containing "syn-thesis block III" shown in Appendix III, plasmid pS 203 containing the entire synthetic gene, and pS 204 repre-senting an expression plasmid which likewise contains the entire synthetic CSF DNA sequence. Expression and acid cleavage result in the same CSF derivative as des-cribed in Figure 2 being obtained.
Figure 11 and its continuation Figure 11a 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 Ile 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 Ile in position 36 and by Leu in positions 46, 79 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 Ile, and Met in posi-tion 46 has been replaced by Leu, and a single Leu residue is present in place of amino acids 79 and 80.
134t19~
Figure 15 shows a synthetic DNA whose use in the synthesis scheme shown in Figure 13 permits the preparation of a CSF derivative in which Met in posi-tion 36 has been replaced by Ile and in position 46 has been replaced by Leu, and in which the two amino acids in positions 79 and 80 have been deleted.
Figure 16 shows synthesis block 1 containing 6 DNA part sequenc es (Ia/Ib, Ic/Id, Ie/If, Ig/Ih, Ii/Ik, I1/Im). The compatible ends of the part-sequences are indicated by angular lines within the DNA-sequence. The numbering above the upper strand of the DNA refers to the nucleotides, the numbering in brackets below the protein sequence refers to the amino acids.
Figure 17 shows synthesis block II containing 3 DNA part-sequences (IIa/IIb, IIc/IId, IIe/IIf). Symbols and numbering see legend of Figure 16. The numbering of synthesis block II is a continuation of the numbering of synthesis block I.
Figure 18 shows synthesis block III containing 6 DNA part-sequences (IIIa/IIIb, IIIc/IIId, IIIe/IIIf, IIIg/IIIh, IIIi/III~, IIIk/III1). Symbols and numbering see legend of Figure 16. The numbering of synthesis block III is a continuation of the numbering of synthesis block II.
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 man-ner known per se to use other protein sequences, especially bacterial, as the "ballast" portion of the fusion proteins, and it is possible to use all custom-ary methods for the linkage and cleavage of the fusion proteins, it being possible for other CSF derivatives with a modified amino acid sequence in the molecule or 'C
-- - Sa -at both ends of the molecule to result. The choice of the IL-2 sequence and the synthetic DNA 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 knovn per se.
It has emerged that the "open reading frame" comprising a ONA 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 prepar-ation 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 are sparingly soluble or insoluble and thus can easily be ~C
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 N-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 elimi-nation of the desired protein.
The natural DNA sequence coding for human IL-2 is dis-closed in the European Patent Application with the pub-lication number~"0,091,539. The literature quoted there also relates to mouse and rat IL-2. These mammalian DNAs can be used for the synthesis of the proteins ac-cording to the invention. However, it is more appro-priate to start from a synthetic ONA, particularly ad-vantageously from the DNA for human IL-2 which has been described in German Offenlegungsschrift 3,419,995 and in the EP-A 0,163,249. 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 fre-quently, E. 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 inven-tion. 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 cleav-age sites.
*in the following text "EP-A"
If use is made 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 S 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 pro-duce as little "ballast" 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 "ballast" portion can be "tailored" to re-,. quirements. Of course, it is also possible to use the DNA sequence - more or less - up to the end and thus generate biologically active IL-2 - modified where appropriate - as "by-product".
Thus the invention relates to fusion proteins of the general formula Met - X - Y - Z or Met - Z - Y - X
(Ia) (Ib) in which X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, prefer-ably, human IL-2, Y denotes a direct bond in the case where the amino acid or amino acid sequence adjacent to the desired protein allows splitting off of the desired protein, or else denotes a bridge member which is com-posed of one or more genetically codable amino acids and allows the splitting, and Z is a sequence of genetically codable amino acids representing the desired CSF pro-tein.
As is evident from formulae Ia and Ib - and as already mentioned above too - it is possible to effect expres-sion of the desired protein in front of or behind the IL-2 portion. In order to simplify, hereinafter 1341197 ' _8_ essentially the second option, which corresponds to the conventional method for the preparation of fusion proteins, will be explained. Thus, although this "classic" variant is described heretofore and herein-after, this is not intended to rule out the other alternative.
The cleavage of the fusion protein can be carried out chemically or enzymatically in a manner known per se.
The choice of the suitable method depends, in particu-lar, on the amino acid sequence of the desired protein.
If there is tryptophan or methionine at the carboxyl terminal end of the bridge member Y, or if Y represents Trp or Met, then chemical cleavage with N-bromosuccin-imide or cyanogen halide can be carried out in the cases where the particular C5F derivatives which are synthe-sized 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 N-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)n-Pro or Glu-(Asp)n-Pro, n denoting 1 to 3.
Examples for enzymatic cleavages are likewise known, it also being possible to use modified enzymes having im-proved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297).
The 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 of the varieties Strepto-myces, B. subtilis, Salmonella typhimurium or Serratia marcescens, in particular E. 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 for this to select the promoter and operator from the group comprising trp, lac, tac, P~ or PR of phage ~, hsp, omp or a synthetic promoter as proposed in, for example, German Offenlegungsschrift 3,430,683 or EP-A 0,173,149. The tac promoter-operator sequence is advantageous, and this is now commercially available (for example expression vector pKK223-3, Pharmacia, "Molecular Biologi~cals, 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 inven-tion likewise relates to them.
Particularly advantageous CSF derivatives are those containing N-terminal proline, since proteins of this type are more stable to attack by proteases. The CSF
derivative which has the entire CSF amino acid se-quence following the proline added to the N-terminal end is particularly preferred. However, it has emerged, surprisingly, that the variants of the CSF
- 10 - 13 4 1 1 9 ~
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, advan-tageously twice or three times. By their nature, the ballast portion in these fusion proteins is reduced, and thus the yield of the desired protein is increased.
The plasmid pHG 23 which was obtained by incorporation of the CSF cDNA sequence into the Pst I cleavage site of pBR 322 has been deposited, in E. coli, at the American Type Culture Collection under number ATCC
39900. The DNA sequence of this corresponds to the variant described in Figure 3 (B) of Wong 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 intro-duced 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 ATG.
The hybrid plasmid (4> is opened with Nco I, and the protruding ends are filled in to give the blunt-ended fragment (5). The vector pUC 12 is opened with the 1341197 .
enzyme Eco RI, whereupon the protruding ends are filled in. This is followed by treatment with bovine alkaline phosphatase, the pUC 12 derivative (6) being obtained.
Ligation of the fragments (5) and (6) results in vec-tons which contain the CSF DNA sequence in both orienta-tions being obtained. They are called pW 203 (7).
Using Eco RI and Rsa I on the vector (7> results in isolation of the fragment (8) which contains the codons for amino acids 63 to 127 of CSF. On the other hand, cutting the vector (4) with Nco I and Rsa I results in isolation of the fragment (9) which contains the codons for amino acids 1 to 61 of CSF.
The plasmid pH 131/5 (German Offenlegungsschrift 3,514,113 or EP-A 0,198,415, Example 1, Figure 1) (10) is cut with Pvu II, the small fragment is removed, and the larger one is ligated to give the 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 Nco I and Eco RI, and the large fragment (12> is isolated.
The fragments (8), (9) and (12) are now ligated to give the hybrid plasmid pW 206 (13). This restores the codon for amino acid 62.
The commercially available plasmid pKK 65-10 (PL Bio-chemical Inc.) is cleaved with Eco RI, and the fragment (14) which contains the two terminators T1 and T2 is isolated. This fragment (14> is inserted into the plasmid (13) which has been opened with Eco RI, the plasmid pW 225 (15) being obtained.
E. coli 24 bacteria which contain the plasmid (15) are cultured in LB medium (J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 30 to 50 ug/ml ampicillin at 37°C
overnight. The culture is diluted in the ratio 1:100 with M9 medium (J. M. Miller, op. cit.) which contains 200 Nm/l casamino acids and 1 Ng/l thiamine, and the mixture is incubated at 37°C with continuous agitation.
At an OD600 = 0.5 or 1 indolyl-3-acrylic acid is added to a final concentration of 15 Ng/l, and the mix-ture is incubated for 2 to 3 hours or 16 hours respect-ively. The bacteria are then removed by centrifug-ation. The bacteria are boiled for five minutes in a buffer mixture (7M urea, 0.1% SDS, 0.1 M sodium phos-phate, 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 con-tains a new protein, in the range of about 14,000-18,000 dalton, which is not found with non-induced cells.
The induction conditions which have been indicated apply to shake cultures; for larger fermentations appropriately modified OD values and, where appropri-ate, slight variations in the inducer concentrations are advantageous.
Example 2 ProO-CSF
The vector pUC 12 is opened with Eco RI and Pst I, and the large fragment C16) 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 lig-ation mixture, and the desired clones which contain the plasmid pW 212 (18) are selected.
The 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 - 13 _ 134 1 1g 7 ' the pUC 8 polylinker (Amann et al., Gene 25 (1983) 167;
EP-A 0,133,282) 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 by by exonuclease treatment in a manner known per se. Ligation results in the plasmid pEW 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 restric-tion enzymes EcoR I and Sal I, and the fragment (22) is isolated.
The plasmid p159/6 (23>, prepared as described in German Offenlegungsschrift 3,419,995 (EP-A 0,163,249), Example 4 (Figure 5), is opened with the restriction enzymes Eco RI
and Sal 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 part-sequence is denoted "~IL2" 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 the fragments (19>, (26) and (27), trans-formation of competent E. coli 294 cells, and selection results in clones which contain the plasmid pW 216 (28) being obtained. The plasmid DNA is characterized by restriction analysis and DNA sequence analysis.
An overnight culture of E. coli cells which contain the plasmid (28) is diluted with LB medium (J. H. Milter, op. cit.), which contains SO Ng/ml ampicillin, in the ratio of about 1:100, and the growth is followed via measurement of the 00. At OD - 0.5, the culture is ad-justed to 1 mM in isopropyl s-galactopyranoside (IPTG) and, after 150 to 180 minutes, the bacteria are removed by centrifugation. The bacteria are boiled for five minutes in a buffer mixture (7M urea, 0.1X SDS, 0.1 M
sodium phosphate, pH 7.0>, and samples are applied to an SDS gel electrophoresis plate. Following electro-phoresis, a protein band which corresponds to the size of the expected fusion protein is obtained from bac-teria which contain the plasmid (28). After disrup-tion of the bacteria (French press; (R)Dyno mill) and centrifugation, the fusion protein is located in the sediment so that it is possible already to remove con-siderable amounts of the other proteins with the super-natant. 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 appropri-ate, slight variations in the IPTG concentrations are advantageous.
1341197 ' Example 3 Pro1-CSF(2-127) Ligation of the fragments (2) (Figure 1) and (16) (Fig-ure 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 pW 240 (32) being obtained. Ex-pression in E. coli, which is carried out as in Example 2, provides a CSF derivative in which the first amino acid has been replaced by proline. This derivative also shows biological activity.
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 nuc-leotide A is eliminated using S1 nuclease, and then the fragment (35) is cut out with Pst I.
Ligation of the 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 - 1b -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 CSF derivative missing the first amino acid. This derivative is bio-logically active.
Example 5 CSF(6-127>
The plasmid (33) (or a corresponding plasmid which con-tains the CSF DNA sequence) is first totally cleaved with Pst I and then partially cleaved with Bst 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 (40) and the fragment (16) (figure 2), 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 DNA sequence for the CSF derivative. This fragment (44) is ligated with the fragments (26) and (27), 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 DNA sequence (46), and thereafter the resulting DNA 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 DNA
sequence for the CSF derivative. Ligation of the frag-ments (49), (26) and (27) provides the hybrid plasmid pW
243 (SO) which corresponds to the plasmid (45) apart from the shortened DNA sequence for the CSF derivative.
Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSF derivative missing the first seven amino acids. This derivative is also biologically active.
Example 7 CSF(12-127) When the synthetic DNA sequence (51) is ligated with the fragments (33) and (16) then the 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 frag-ment (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 ProO-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 cor-responding plasmid which contains the CSF DNA sequence) first with Pst I and then partially with Bst NI results in the DNA sequence (56) which comprises the largest part of the CSF sequence being obtained.
The 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 linea-rized plasmid (58) is ligated with the fragments (56) and (57), then the hybrid plasmid pW 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 (60) which contains the modified CSF DNA
sequence. This fragment (60) is ligated with the syn-thetic DNA sequence (61) and the fragment (55), the DNA sequence (62) being obtained. The latter is lig-ated with the DNA fragments (26) and (27) (Figure 2), the hybrid plasmid pW 246 (b3> being obtained. This plasmid is shown twice in Figure 8a, the lower repre-sentation 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
derivative which is extended by an N-terminal proline and in which, additionally, the final amino acid has been replaced by aspartic acid. This derivative is biologically active.
Example 9 ProO-CSF(1-126)-Asp The hybrid plasmid (63) (Figure 8) is cleaved with Eco RI and Pst I, and the fragment which contains the 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 iso-lated. The fragment (66) is cleared with Bst NI, and the fragment (67) is isolated. Ligation of the DNA se-quences (27), (65), (67), (61) and (60) results in the hybrid plasmid pW 247 (68) in 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 as in Example 8.
Example 10 Synthetic gene (for ProO-CSF) Processes known per se, for example the phosphite method (German Offenlegungsschriften 3,327,007, 3,328,793, 3,409,9b6, 3,414,831 and 3,419,995) are used to synthesize the three "synthesis blocks" I (CSF-I), designated (69) in the figures, II (CSF-II), (70) in the figures, and III (CSF-III), (71) in the figures.
The synthesized oligonucleotides Ia to Im, IIa to IIf and IIIa to IIII are indicated in the nucleotide 25 sequence of these synthesis blocks (Appendix).
The choice of the nucleotides for the synthetic gene _ Zo _ 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 known per se, to exchange, add, or delete codons for amino acids.
Synthesis Block I (CSF I) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) -- Nar I ('rG+CGCC 1 Hpa II C+CGG 4 Fiae II GGCGC+C 4 Nae I GCC+GGC 5 Pvu I CGAT+CG 13 Sal I G+TCGAC 24 Acc I GT+CGAC 25 Hinc II GTC+GAC 26 Hpa I/ GTT+AAC 48 Ainc II
Hha I GCG+C 66 Hint I G+AGTC 88 Nru I TCG+CGA 89 Xma III C+GGCCG 95 Sac II CCGC+CG
Eco R0 GAT+ATC 128 Synthesis Block II (CSF-II) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) AtlIII A+CATGT
MluI A+CGCGT
XhoI C+TCGAC ~?5 TaqI T+CGA ~~6 Synthesis Block I-I (CSF-II) (cont.) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) Hga I GACGC (5/10) ~~~
Ava I C+TCGAG 177 Alu I . AG+CT .180 Sac I/ GAGCT+C 182 Hgi AI
Stu I/ AGG+CCT 194 Hae I
Synthesis Block III (CSF-III) Enzyme Cut after nucleotide Recognition sequence no. (coding strand) A~1 II C+TTAAG 217 Hae III GG+CC 224 Apa I GGGCC+C 22'7 Mnl I CCTC (7/7) 238 Nhe I G+CTAGC 241 Mae I C+TAG 242 Aha II GA+CGTC 280 Aat II GACGT+C 283 Sci NI G+CGC 287 Mst I TCG+GCA 288 Sau 3AI/ +GATC 296 Mbo I
Dpn I GA+TC 298 Asu II TT+CGAA 308 Aha III TTT+AAA 318 Ava II G+GTCC 382 Eco RII +CCAGG 384 Est NI/ CC+AGG 380 Scr FI
The three synthesis blocks were first individually cloned, amplified in E. coli and re-isolated:
Synthesis block CSF-I (69) is incorporated in the pUC
12 derivative (16), the plasmid pS 200 (72) being obtained.
pUC 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 obtained.
pUC 13 is opened with Hind III and Sma I, and the lin-earized plasmid (75> is ligated with CSF-III (71>, the 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 linearized with Eco RI 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 Bam HI, and 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 the bridge member which per mits 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 the N-terminal end.
134119?
Example 11 CSF(1-12)His(14-121)His(123-127) When the nucleotides in synthesis block I up to No. 48 (cleavage site for Hpa I) 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 in which there is Trp in front of the first amino acid (Ala), and Trp in position 13 has been replaced by His.
The plasmid (72) (Figure 10) is opened with Eco RI and Hpa 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 CSF I (CSF I') being obtained.
The plasmid (76) (Figure 10) is opened with Hind III
and Sal I, and the small (86) and large (87) fragments are isolated. The small fragment (86) is then cut with Taq I, and the fragment (88) is isolated.
The 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 E. 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
I' is isolated.
When 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 part-sequence 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 His, 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 histidine.
Example 12 CSF(1-99)Thr(101-127) When, in the synthesis of the synthesis block III, oligonucleotides IIIe and IIIf are replaced by the synthetic sequence (94) and the process is otherwise carried out as in Example 10, then a CSF derivative in which Ile in position 100 has been replaced by Thr is obtained.
Example 13 CSF(1-35>Ile(37-45>Leu(47-78)Leu-Leu(81-127) First the oligonucleotide (95) which contains in posi-tion 36 the codon for Ile 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 III, and the fragment (97) is isolated.
In addition, the sequence (98) in which the codon for Met is located in front of that for the first amino acid is synthesized.
When the fragments (16), (98), (97), (95) and (96> are now ligated then the plasmid pS 208 (99) is obtained.
This corresponds to the plasmid (72) but contains in 134119' position 0 of the CSF I sequence the codon for Met, in position 36 a codon for Ile, 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 Hind 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 posi-tions 79 and 80 in the CSF III sequence is obtained.
The plasmid (93) (Figure 11a) is now partially cut with Pvu I and with Sal I, ahd the large fragment (103) is isolated. The plasmid (99) is likewise partially opened with Pvu I and with Pst I, and the small frag-ment (104), which contains the modified CSF I sequence is isolated. In addition, the plasmid (102) is opened with Hind 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 11a) 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 the other amino acids.
When E. coli is transformed with the plasmid (106) then, after inductian, a fusion protein is obtained which can be cleaved with cyanogen halide resulting in a CSF derivative which contains Ile 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 Ile in position 36 and Leu in position 46, and in which the amino acid Leu is present in place of amino acids 79 and 80, is obtained.
Example 15 CSF(1-35)Ile(37-45)Leu(47-78)-(81-127) When the process is carried out as in Example 13 but the synthetic sequence (108) is used in place of the syn-thetic sequence (100), then a deletion product which' has Ile in position 36 and Leu in position 46, and in which the amino acids in positions 79 and 80 have been deleted, is obtained.
1~411g~
APPENDIX
Synthesis block I (CSF I) (69) Ic AAT TCG ATC GAC GAC CCG GCG CCG GCC CGA TCG CCG TCT CCG
GC TAG CTG CTG GGC CGC GGC CGG GCT AGC GGC AGA GGC
(Eco RI) Ile Asp Asp Pro Ala Pro Ala Arg Ser Pro Ser Pro b (,) (5) ,~, Z
.r- - le 50 Z
Ic ~
.
TCG ACC CAG CCC TGG GAA CAC GTT AAC GCG ATC CAG G GCG
AGC TGG GTC GGG ACC CTT GTG CAA TTG CGC TAG GTC CTT CGC
Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala t5 (,o) c~5) (20) ~ f .
d r ,oo .
CGG CGT CTG CTG AAC CTG AGT CGC GAC ACG GCC GAA ATG
GCG
20 G(' GCA GAC GAC TTG GAC TCA GCG CTG TGC CGG CTT TAC
CGC
Arg Arg ?~eu Asn Zeu Ser Arg Asp Thr Ala Glu Met Zeu Ala (25) __~ (30) (35) .I k ~t _ ~
25 . . .
~
AAC GAA ACC GTT GAA GTG ATA GAG ATG TTC GAC CTG CA
TCT
TTG CTT TGG CAA CTT CAC TAT CTC TAC AAG CTG G
AGA (Pst I) Asn Glu Thr Val Glu Val Ile Glu Met Phe Asp(Zeu) Ser (40) (45) (50) 3 Im 13 4 1'~9~ .
_za_ Synthesis block II (CSF II) (70>
~ 5 0 a ,.
Jl G
(Pst I)G CCG ACA TGT CTC CAG ACG CGT CTC GAG CTC TAC
GAA
AC GTC CTT GGC TGT ACA GAG GTC TGC CTC GAG ATrr GCA
GAG
(Gln)('rlu Pro Thr Cps Zeu Gln Thr Glu Leu Tar Arg Zeu (50) (55) (60) _ _ LL 6 ( I
~ ~
. ,~
c yc j~'e ~.
AAA CAA GGC CTT CGT GGT CTG ACC A (Hind III) TCT
TTT GTT CCG CCA GAC TGG TTC GA
GAA AGA
GCA
Las Gln Gly Gly Zeu Thr(Z~s) heu Ser Arg (65) (?0) I( --,.
' 1341 19~
Synthesis block III (CSF III) (71) a ~ -.- -. . . . .
AG CTT AAG GGG CCCCTC ACC ATG ATG GCT AGC CACTAC AAA
(Hind III)A TTC CCC GGGGAG TGG TAC TAC CGA TCG GTGATG ~_'TT
( Leu ) Gly ProLeu Thr Met Met Ala Ser HisTyr Lys , Lys (72) (75) (80 ) (85) ,b ~
' '' IZ
a CAG CAC TGC CCG CCGACT CCG GAG ACG TCT TGC GCAACG CAG
GTC GTG ACG GGC GGCTGA GGC CTC TGC AGA ACG CGTTGC GTC
Gln His Cys Pro ProThr Pro Glu Thr Ser Cys AlaThr Gln (90)~ (95) ~
. . ~~
.
ATC ATC ACC TTC GAATCT TTT AAA GAA AAC CTG AAGGAC TTT
TAG TAG TGG AAG CTTAGA AAA TTT CTT TTG GAC TTCCTG AAA
Ile Ile Thr Phe GluSer Phe Lys Glu Asn Leu LysAsp Phe (100) ( 105) ( 110) ~~
350 ~ , ~ 391 ~ z . ; ~ ., r--~' k 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 Gln Glu (115) (120) (125) X11 ~C
Sal I Pst I
TGA TAG T~T GC GCC C
ACT ATC AGC TGA CGT CGG G
Stp Stp (Sma I)
It has now been found, surprisingly, that the expres-sion of human GM-CSF protein, called "CSF" hereinafter, in bacteria results in a biologically active product.
Thus the invention relates to CSF for use in medical treatment and to the use for the preparation of medi-cements.
The invention furthermore relates to the preparation of CSF by expression in bacteria, in particular in E.
coli. In particular, it is possible to use for this purpose the published cDNA sequences which can be ob-tained in a manner known per se, preferably by synthe-sis.
The invention additionally relates to expression vectors for use in bacteria, in particular in E. coli, which contain, in a suitable arrangement ("operatively linked to">, a DNA coding for CSF or a CSF fusion pro-tein.
The invention additionally relates to biologically active derivatives of CSF which can be obtained by modifications, which are known per se, of the ONA
sequences. Thus, for example, it is possible to in-corporate cleavage sites in the construction of ~3'~~19~
vectors for fusion proteins which, after elimination of the CSF protein, have C-terminal and N-terminal modifi-cations in the amino acid sequence. Furthermore, the invention relates to the use of proteins of this type in medical treatment and to their use for the prepara-tion of medicaments, and to medicaments which contain CSF protein and its biologically active derivatives, in particular medicaments for the stimulation of prolifer-ation of hemopoietic cells and for promotion of the formation of granulocytes and macrophages.
Further aspects of the invention and its preferred em-bodiments are illustrated in detail below and are defined in the patent claims.
The 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 the same numbers. These figures are not to scale, in particular the scale has been "expanded" in the region of the polylinkers.
Thus, Figure 1 and its continuations 1a and 1b show the preparation of the vector pW 225 which is used for the direct expression of (Met-)CSF. The figures which fol-low relate to vectors which result in the expression of fusion proteins in which a "ballast" protein, which is derived from a part-sequence of human interleukin-2, hereinafter "IL-2" or "eIL-2", is located at the N-terminal end in front of the CSF amino acid sequence:
Figure 2 and its continuations 2a and 2b show the prep-aration 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 pW 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 pW 241 which codes for a fusion protein which results, after S acid cleavage, in 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 acids have been eliminated.
Figure 6 relates to the 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 in a CSF
derivative in which proline is located at the N-termi-nal end in 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' sequen-ces 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 pre-paration of the hybrid plasmids pS 200 to 204 which contain synthetic CSF DNA part-sequences, the plasmid pS 200 containing "synthesis block I", shown in Appen-dix I, plasmid pS 201 containing "synthesis block II"
shown in Appendix II, plasmid pS 202 containing "syn-thesis block III" shown in Appendix III, plasmid pS 203 containing the entire synthetic gene, and pS 204 repre-senting an expression plasmid which likewise contains the entire synthetic CSF DNA sequence. Expression and acid cleavage result in the same CSF derivative as des-cribed in Figure 2 being obtained.
Figure 11 and its continuation Figure 11a 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 Ile 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 Ile in position 36 and by Leu in positions 46, 79 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 Ile, and Met in posi-tion 46 has been replaced by Leu, and a single Leu residue is present in place of amino acids 79 and 80.
134t19~
Figure 15 shows a synthetic DNA whose use in the synthesis scheme shown in Figure 13 permits the preparation of a CSF derivative in which Met in posi-tion 36 has been replaced by Ile and in position 46 has been replaced by Leu, and in which the two amino acids in positions 79 and 80 have been deleted.
Figure 16 shows synthesis block 1 containing 6 DNA part sequenc es (Ia/Ib, Ic/Id, Ie/If, Ig/Ih, Ii/Ik, I1/Im). The compatible ends of the part-sequences are indicated by angular lines within the DNA-sequence. The numbering above the upper strand of the DNA refers to the nucleotides, the numbering in brackets below the protein sequence refers to the amino acids.
Figure 17 shows synthesis block II containing 3 DNA part-sequences (IIa/IIb, IIc/IId, IIe/IIf). Symbols and numbering see legend of Figure 16. The numbering of synthesis block II is a continuation of the numbering of synthesis block I.
Figure 18 shows synthesis block III containing 6 DNA part-sequences (IIIa/IIIb, IIIc/IIId, IIIe/IIIf, IIIg/IIIh, IIIi/III~, IIIk/III1). Symbols and numbering see legend of Figure 16. The numbering of synthesis block III is a continuation of the numbering of synthesis block II.
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 man-ner known per se to use other protein sequences, especially bacterial, as the "ballast" portion of the fusion proteins, and it is possible to use all custom-ary methods for the linkage and cleavage of the fusion proteins, it being possible for other CSF derivatives with a modified amino acid sequence in the molecule or 'C
-- - Sa -at both ends of the molecule to result. The choice of the IL-2 sequence and the synthetic DNA 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 knovn per se.
It has emerged that the "open reading frame" comprising a ONA 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 prepar-ation 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 are sparingly soluble or insoluble and thus can easily be ~C
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 N-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 elimi-nation of the desired protein.
The natural DNA sequence coding for human IL-2 is dis-closed in the European Patent Application with the pub-lication number~"0,091,539. The literature quoted there also relates to mouse and rat IL-2. These mammalian DNAs can be used for the synthesis of the proteins ac-cording to the invention. However, it is more appro-priate to start from a synthetic ONA, particularly ad-vantageously from the DNA for human IL-2 which has been described in German Offenlegungsschrift 3,419,995 and in the EP-A 0,163,249. 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 fre-quently, E. 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 inven-tion. 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 cleav-age sites.
*in the following text "EP-A"
If use is made 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 S 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 pro-duce as little "ballast" 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 "ballast" portion can be "tailored" to re-,. quirements. Of course, it is also possible to use the DNA sequence - more or less - up to the end and thus generate biologically active IL-2 - modified where appropriate - as "by-product".
Thus the invention relates to fusion proteins of the general formula Met - X - Y - Z or Met - Z - Y - X
(Ia) (Ib) in which X essentially denotes the amino acid sequence of approximately the first 100 amino acids of, prefer-ably, human IL-2, Y denotes a direct bond in the case where the amino acid or amino acid sequence adjacent to the desired protein allows splitting off of the desired protein, or else denotes a bridge member which is com-posed of one or more genetically codable amino acids and allows the splitting, and Z is a sequence of genetically codable amino acids representing the desired CSF pro-tein.
As is evident from formulae Ia and Ib - and as already mentioned above too - it is possible to effect expres-sion of the desired protein in front of or behind the IL-2 portion. In order to simplify, hereinafter 1341197 ' _8_ essentially the second option, which corresponds to the conventional method for the preparation of fusion proteins, will be explained. Thus, although this "classic" variant is described heretofore and herein-after, this is not intended to rule out the other alternative.
The cleavage of the fusion protein can be carried out chemically or enzymatically in a manner known per se.
The choice of the suitable method depends, in particu-lar, on the amino acid sequence of the desired protein.
If there is tryptophan or methionine at the carboxyl terminal end of the bridge member Y, or if Y represents Trp or Met, then chemical cleavage with N-bromosuccin-imide or cyanogen halide can be carried out in the cases where the particular C5F derivatives which are synthe-sized 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 N-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)n-Pro or Glu-(Asp)n-Pro, n denoting 1 to 3.
Examples for enzymatic cleavages are likewise known, it also being possible to use modified enzymes having im-proved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297).
The 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 of the varieties Strepto-myces, B. subtilis, Salmonella typhimurium or Serratia marcescens, in particular E. 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 for this to select the promoter and operator from the group comprising trp, lac, tac, P~ or PR of phage ~, hsp, omp or a synthetic promoter as proposed in, for example, German Offenlegungsschrift 3,430,683 or EP-A 0,173,149. The tac promoter-operator sequence is advantageous, and this is now commercially available (for example expression vector pKK223-3, Pharmacia, "Molecular Biologi~cals, 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 inven-tion likewise relates to them.
Particularly advantageous CSF derivatives are those containing N-terminal proline, since proteins of this type are more stable to attack by proteases. The CSF
derivative which has the entire CSF amino acid se-quence following the proline added to the N-terminal end is particularly preferred. However, it has emerged, surprisingly, that the variants of the CSF
- 10 - 13 4 1 1 9 ~
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, advan-tageously twice or three times. By their nature, the ballast portion in these fusion proteins is reduced, and thus the yield of the desired protein is increased.
The plasmid pHG 23 which was obtained by incorporation of the CSF cDNA sequence into the Pst I cleavage site of pBR 322 has been deposited, in E. coli, at the American Type Culture Collection under number ATCC
39900. The DNA sequence of this corresponds to the variant described in Figure 3 (B) of Wong 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 intro-duced 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 ATG.
The hybrid plasmid (4> is opened with Nco I, and the protruding ends are filled in to give the blunt-ended fragment (5). The vector pUC 12 is opened with the 1341197 .
enzyme Eco RI, whereupon the protruding ends are filled in. This is followed by treatment with bovine alkaline phosphatase, the pUC 12 derivative (6) being obtained.
Ligation of the fragments (5) and (6) results in vec-tons which contain the CSF DNA sequence in both orienta-tions being obtained. They are called pW 203 (7).
Using Eco RI and Rsa I on the vector (7> results in isolation of the fragment (8) which contains the codons for amino acids 63 to 127 of CSF. On the other hand, cutting the vector (4) with Nco I and Rsa I results in isolation of the fragment (9) which contains the codons for amino acids 1 to 61 of CSF.
The plasmid pH 131/5 (German Offenlegungsschrift 3,514,113 or EP-A 0,198,415, Example 1, Figure 1) (10) is cut with Pvu II, the small fragment is removed, and the larger one is ligated to give the 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 Nco I and Eco RI, and the large fragment (12> is isolated.
The fragments (8), (9) and (12) are now ligated to give the hybrid plasmid pW 206 (13). This restores the codon for amino acid 62.
The commercially available plasmid pKK 65-10 (PL Bio-chemical Inc.) is cleaved with Eco RI, and the fragment (14) which contains the two terminators T1 and T2 is isolated. This fragment (14> is inserted into the plasmid (13) which has been opened with Eco RI, the plasmid pW 225 (15) being obtained.
E. coli 24 bacteria which contain the plasmid (15) are cultured in LB medium (J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) containing 30 to 50 ug/ml ampicillin at 37°C
overnight. The culture is diluted in the ratio 1:100 with M9 medium (J. M. Miller, op. cit.) which contains 200 Nm/l casamino acids and 1 Ng/l thiamine, and the mixture is incubated at 37°C with continuous agitation.
At an OD600 = 0.5 or 1 indolyl-3-acrylic acid is added to a final concentration of 15 Ng/l, and the mix-ture is incubated for 2 to 3 hours or 16 hours respect-ively. The bacteria are then removed by centrifug-ation. The bacteria are boiled for five minutes in a buffer mixture (7M urea, 0.1% SDS, 0.1 M sodium phos-phate, 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 con-tains a new protein, in the range of about 14,000-18,000 dalton, which is not found with non-induced cells.
The induction conditions which have been indicated apply to shake cultures; for larger fermentations appropriately modified OD values and, where appropri-ate, slight variations in the inducer concentrations are advantageous.
Example 2 ProO-CSF
The vector pUC 12 is opened with Eco RI and Pst I, and the large fragment C16) 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 lig-ation mixture, and the desired clones which contain the plasmid pW 212 (18) are selected.
The 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 - 13 _ 134 1 1g 7 ' the pUC 8 polylinker (Amann et al., Gene 25 (1983) 167;
EP-A 0,133,282) 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 by by exonuclease treatment in a manner known per se. Ligation results in the plasmid pEW 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 restric-tion enzymes EcoR I and Sal I, and the fragment (22) is isolated.
The plasmid p159/6 (23>, prepared as described in German Offenlegungsschrift 3,419,995 (EP-A 0,163,249), Example 4 (Figure 5), is opened with the restriction enzymes Eco RI
and Sal 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 part-sequence is denoted "~IL2" 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 the fragments (19>, (26) and (27), trans-formation of competent E. coli 294 cells, and selection results in clones which contain the plasmid pW 216 (28) being obtained. The plasmid DNA is characterized by restriction analysis and DNA sequence analysis.
An overnight culture of E. coli cells which contain the plasmid (28) is diluted with LB medium (J. H. Milter, op. cit.), which contains SO Ng/ml ampicillin, in the ratio of about 1:100, and the growth is followed via measurement of the 00. At OD - 0.5, the culture is ad-justed to 1 mM in isopropyl s-galactopyranoside (IPTG) and, after 150 to 180 minutes, the bacteria are removed by centrifugation. The bacteria are boiled for five minutes in a buffer mixture (7M urea, 0.1X SDS, 0.1 M
sodium phosphate, pH 7.0>, and samples are applied to an SDS gel electrophoresis plate. Following electro-phoresis, a protein band which corresponds to the size of the expected fusion protein is obtained from bac-teria which contain the plasmid (28). After disrup-tion of the bacteria (French press; (R)Dyno mill) and centrifugation, the fusion protein is located in the sediment so that it is possible already to remove con-siderable amounts of the other proteins with the super-natant. 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 appropri-ate, slight variations in the IPTG concentrations are advantageous.
1341197 ' Example 3 Pro1-CSF(2-127) Ligation of the fragments (2) (Figure 1) and (16) (Fig-ure 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 pW 240 (32) being obtained. Ex-pression in E. coli, which is carried out as in Example 2, provides a CSF derivative in which the first amino acid has been replaced by proline. This derivative also shows biological activity.
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 nuc-leotide A is eliminated using S1 nuclease, and then the fragment (35) is cut out with Pst I.
Ligation of the 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 - 1b -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 CSF derivative missing the first amino acid. This derivative is bio-logically active.
Example 5 CSF(6-127>
The plasmid (33) (or a corresponding plasmid which con-tains the CSF DNA sequence) is first totally cleaved with Pst I and then partially cleaved with Bst 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 (40) and the fragment (16) (figure 2), 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 DNA sequence for the CSF derivative. This fragment (44) is ligated with the fragments (26) and (27), 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 DNA sequence (46), and thereafter the resulting DNA 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 DNA
sequence for the CSF derivative. Ligation of the frag-ments (49), (26) and (27) provides the hybrid plasmid pW
243 (SO) which corresponds to the plasmid (45) apart from the shortened DNA sequence for the CSF derivative.
Expression as in Example 2 results in a fusion protein which, after acid cleavage, provides a CSF derivative missing the first seven amino acids. This derivative is also biologically active.
Example 7 CSF(12-127) When the synthetic DNA sequence (51) is ligated with the fragments (33) and (16) then the 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 frag-ment (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 ProO-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 cor-responding plasmid which contains the CSF DNA sequence) first with Pst I and then partially with Bst NI results in the DNA sequence (56) which comprises the largest part of the CSF sequence being obtained.
The 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 linea-rized plasmid (58) is ligated with the fragments (56) and (57), then the hybrid plasmid pW 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 (60) which contains the modified CSF DNA
sequence. This fragment (60) is ligated with the syn-thetic DNA sequence (61) and the fragment (55), the DNA sequence (62) being obtained. The latter is lig-ated with the DNA fragments (26) and (27) (Figure 2), the hybrid plasmid pW 246 (b3> being obtained. This plasmid is shown twice in Figure 8a, the lower repre-sentation 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
derivative which is extended by an N-terminal proline and in which, additionally, the final amino acid has been replaced by aspartic acid. This derivative is biologically active.
Example 9 ProO-CSF(1-126)-Asp The hybrid plasmid (63) (Figure 8) is cleaved with Eco RI and Pst I, and the fragment which contains the 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 iso-lated. The fragment (66) is cleared with Bst NI, and the fragment (67) is isolated. Ligation of the DNA se-quences (27), (65), (67), (61) and (60) results in the hybrid plasmid pW 247 (68) in 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 as in Example 8.
Example 10 Synthetic gene (for ProO-CSF) Processes known per se, for example the phosphite method (German Offenlegungsschriften 3,327,007, 3,328,793, 3,409,9b6, 3,414,831 and 3,419,995) are used to synthesize the three "synthesis blocks" I (CSF-I), designated (69) in the figures, II (CSF-II), (70) in the figures, and III (CSF-III), (71) in the figures.
The synthesized oligonucleotides Ia to Im, IIa to IIf and IIIa to IIII are indicated in the nucleotide 25 sequence of these synthesis blocks (Appendix).
The choice of the nucleotides for the synthetic gene _ Zo _ 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 known per se, to exchange, add, or delete codons for amino acids.
Synthesis Block I (CSF I) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) -- Nar I ('rG+CGCC 1 Hpa II C+CGG 4 Fiae II GGCGC+C 4 Nae I GCC+GGC 5 Pvu I CGAT+CG 13 Sal I G+TCGAC 24 Acc I GT+CGAC 25 Hinc II GTC+GAC 26 Hpa I/ GTT+AAC 48 Ainc II
Hha I GCG+C 66 Hint I G+AGTC 88 Nru I TCG+CGA 89 Xma III C+GGCCG 95 Sac II CCGC+CG
Eco R0 GAT+ATC 128 Synthesis Block II (CSF-II) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) AtlIII A+CATGT
MluI A+CGCGT
XhoI C+TCGAC ~?5 TaqI T+CGA ~~6 Synthesis Block I-I (CSF-II) (cont.) Enzyme Recognition sequence Cut after nucleotide no. (coding strand) Hga I GACGC (5/10) ~~~
Ava I C+TCGAG 177 Alu I . AG+CT .180 Sac I/ GAGCT+C 182 Hgi AI
Stu I/ AGG+CCT 194 Hae I
Synthesis Block III (CSF-III) Enzyme Cut after nucleotide Recognition sequence no. (coding strand) A~1 II C+TTAAG 217 Hae III GG+CC 224 Apa I GGGCC+C 22'7 Mnl I CCTC (7/7) 238 Nhe I G+CTAGC 241 Mae I C+TAG 242 Aha II GA+CGTC 280 Aat II GACGT+C 283 Sci NI G+CGC 287 Mst I TCG+GCA 288 Sau 3AI/ +GATC 296 Mbo I
Dpn I GA+TC 298 Asu II TT+CGAA 308 Aha III TTT+AAA 318 Ava II G+GTCC 382 Eco RII +CCAGG 384 Est NI/ CC+AGG 380 Scr FI
The three synthesis blocks were first individually cloned, amplified in E. coli and re-isolated:
Synthesis block CSF-I (69) is incorporated in the pUC
12 derivative (16), the plasmid pS 200 (72) being obtained.
pUC 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 obtained.
pUC 13 is opened with Hind III and Sma I, and the lin-earized plasmid (75> is ligated with CSF-III (71>, the 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 linearized with Eco RI 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 Bam HI, and 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 the bridge member which per mits 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 the N-terminal end.
134119?
Example 11 CSF(1-12)His(14-121)His(123-127) When the nucleotides in synthesis block I up to No. 48 (cleavage site for Hpa I) 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 in which there is Trp in front of the first amino acid (Ala), and Trp in position 13 has been replaced by His.
The plasmid (72) (Figure 10) is opened with Eco RI and Hpa 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 CSF I (CSF I') being obtained.
The plasmid (76) (Figure 10) is opened with Hind III
and Sal I, and the small (86) and large (87) fragments are isolated. The small fragment (86) is then cut with Taq I, and the fragment (88) is isolated.
The 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 E. 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
I' is isolated.
When 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 part-sequence 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 His, 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 histidine.
Example 12 CSF(1-99)Thr(101-127) When, in the synthesis of the synthesis block III, oligonucleotides IIIe and IIIf are replaced by the synthetic sequence (94) and the process is otherwise carried out as in Example 10, then a CSF derivative in which Ile in position 100 has been replaced by Thr is obtained.
Example 13 CSF(1-35>Ile(37-45>Leu(47-78)Leu-Leu(81-127) First the oligonucleotide (95) which contains in posi-tion 36 the codon for Ile 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 III, and the fragment (97) is isolated.
In addition, the sequence (98) in which the codon for Met is located in front of that for the first amino acid is synthesized.
When the fragments (16), (98), (97), (95) and (96> are now ligated then the plasmid pS 208 (99) is obtained.
This corresponds to the plasmid (72) but contains in 134119' position 0 of the CSF I sequence the codon for Met, in position 36 a codon for Ile, 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 Hind 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 posi-tions 79 and 80 in the CSF III sequence is obtained.
The plasmid (93) (Figure 11a) is now partially cut with Pvu I and with Sal I, ahd the large fragment (103) is isolated. The plasmid (99) is likewise partially opened with Pvu I and with Pst I, and the small frag-ment (104), which contains the modified CSF I sequence is isolated. In addition, the plasmid (102) is opened with Hind 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 11a) 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 the other amino acids.
When E. coli is transformed with the plasmid (106) then, after inductian, a fusion protein is obtained which can be cleaved with cyanogen halide resulting in a CSF derivative which contains Ile 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 Ile in position 36 and Leu in position 46, and in which the amino acid Leu is present in place of amino acids 79 and 80, is obtained.
Example 15 CSF(1-35)Ile(37-45)Leu(47-78)-(81-127) When the process is carried out as in Example 13 but the synthetic sequence (108) is used in place of the syn-thetic sequence (100), then a deletion product which' has Ile in position 36 and Leu in position 46, and in which the amino acids in positions 79 and 80 have been deleted, is obtained.
1~411g~
APPENDIX
Synthesis block I (CSF I) (69) Ic AAT TCG ATC GAC GAC CCG GCG CCG GCC CGA TCG CCG TCT CCG
GC TAG CTG CTG GGC CGC GGC CGG GCT AGC GGC AGA GGC
(Eco RI) Ile Asp Asp Pro Ala Pro Ala Arg Ser Pro Ser Pro b (,) (5) ,~, Z
.r- - le 50 Z
Ic ~
.
TCG ACC CAG CCC TGG GAA CAC GTT AAC GCG ATC CAG G GCG
AGC TGG GTC GGG ACC CTT GTG CAA TTG CGC TAG GTC CTT CGC
Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala t5 (,o) c~5) (20) ~ f .
d r ,oo .
CGG CGT CTG CTG AAC CTG AGT CGC GAC ACG GCC GAA ATG
GCG
20 G(' GCA GAC GAC TTG GAC TCA GCG CTG TGC CGG CTT TAC
CGC
Arg Arg ?~eu Asn Zeu Ser Arg Asp Thr Ala Glu Met Zeu Ala (25) __~ (30) (35) .I k ~t _ ~
25 . . .
~
AAC GAA ACC GTT GAA GTG ATA GAG ATG TTC GAC CTG CA
TCT
TTG CTT TGG CAA CTT CAC TAT CTC TAC AAG CTG G
AGA (Pst I) Asn Glu Thr Val Glu Val Ile Glu Met Phe Asp(Zeu) Ser (40) (45) (50) 3 Im 13 4 1'~9~ .
_za_ Synthesis block II (CSF II) (70>
~ 5 0 a ,.
Jl G
(Pst I)G CCG ACA TGT CTC CAG ACG CGT CTC GAG CTC TAC
GAA
AC GTC CTT GGC TGT ACA GAG GTC TGC CTC GAG ATrr GCA
GAG
(Gln)('rlu Pro Thr Cps Zeu Gln Thr Glu Leu Tar Arg Zeu (50) (55) (60) _ _ LL 6 ( I
~ ~
. ,~
c yc j~'e ~.
AAA CAA GGC CTT CGT GGT CTG ACC A (Hind III) TCT
TTT GTT CCG CCA GAC TGG TTC GA
GAA AGA
GCA
Las Gln Gly Gly Zeu Thr(Z~s) heu Ser Arg (65) (?0) I( --,.
' 1341 19~
Synthesis block III (CSF III) (71) a ~ -.- -. . . . .
AG CTT AAG GGG CCCCTC ACC ATG ATG GCT AGC CACTAC AAA
(Hind III)A TTC CCC GGGGAG TGG TAC TAC CGA TCG GTGATG ~_'TT
( Leu ) Gly ProLeu Thr Met Met Ala Ser HisTyr Lys , Lys (72) (75) (80 ) (85) ,b ~
' '' IZ
a CAG CAC TGC CCG CCGACT CCG GAG ACG TCT TGC GCAACG CAG
GTC GTG ACG GGC GGCTGA GGC CTC TGC AGA ACG CGTTGC GTC
Gln His Cys Pro ProThr Pro Glu Thr Ser Cys AlaThr Gln (90)~ (95) ~
. . ~~
.
ATC ATC ACC TTC GAATCT TTT AAA GAA AAC CTG AAGGAC TTT
TAG TAG TGG AAG CTTAGA AAA TTT CTT TTG GAC TTCCTG AAA
Ile Ile Thr Phe GluSer Phe Lys Glu Asn Leu LysAsp Phe (100) ( 105) ( 110) ~~
350 ~ , ~ 391 ~ z . ; ~ ., r--~' k 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 Gln Glu (115) (120) (125) X11 ~C
Sal I Pst I
TGA TAG T~T GC GCC C
ACT ATC AGC TGA CGT CGG G
Stp Stp (Sma I)
Claims (4)
1. A fusion protean of the general formula Ia or Ib Met-X-Y-Z Met-Z-Y-X
(Ia) (Ib) wherein X represents the amino acid sequence of approximately the first 100 amino acids of IL-2, Z is a sequence of genetically codable amino acids representing a GM-CSF protein having an amino acid sequence comprising amino acids 1(Ala) - 49(Leu) as shown in Figure 16, amino acids 50(Gln) - 72(Lys) as shown in Figure 17, and amino acids 73(Leu) - 127(Glu) as shown in Figure 18, and Y
represents a direct bond in the case where the amino acid or amino acid sequence adjacent to the GM-CSF protein allows splitting off of the GM-CSF protein, or else denotes a bridge member which is composed of one or more genetically codable amino acids and allows the splitting.
(Ia) (Ib) wherein X represents the amino acid sequence of approximately the first 100 amino acids of IL-2, Z is a sequence of genetically codable amino acids representing a GM-CSF protein having an amino acid sequence comprising amino acids 1(Ala) - 49(Leu) as shown in Figure 16, amino acids 50(Gln) - 72(Lys) as shown in Figure 17, and amino acids 73(Leu) - 127(Glu) as shown in Figure 18, and Y
represents a direct bond in the case where the amino acid or amino acid sequence adjacent to the GM-CSF protein allows splitting off of the GM-CSF protein, or else denotes a bridge member which is composed of one or more genetically codable amino acids and allows the splitting.
2. A fusion protein of the general formula Ia or Ib Met-X-Y-Z Met-Z-Y-X
(Ia) (Ib) wherein X represents the amino acid sequence of approximately the first 100 amino acids of IL-2, Z as a sequence of genetically codable amino acids representing a GM-CSF protein having the amino acid sequence 1(Ala) - 49(Leu) as shown in Figure 16, amino acids 50(Gln) - 72(Lys) as shown in Figure 17, and amino acids 73(Leu) - 127(Glu) as shown in Figure 18, and Y represents a direct bond in the case where the amino acid or amino acid sequence adjacent to the GM-CSF protein allows splitting off of the GM-CSF protein, or else denotes a bridge member which is composed of one or more genetically codable amino acids and allows the splitting.
(Ia) (Ib) wherein X represents the amino acid sequence of approximately the first 100 amino acids of IL-2, Z as a sequence of genetically codable amino acids representing a GM-CSF protein having the amino acid sequence 1(Ala) - 49(Leu) as shown in Figure 16, amino acids 50(Gln) - 72(Lys) as shown in Figure 17, and amino acids 73(Leu) - 127(Glu) as shown in Figure 18, and Y represents a direct bond in the case where the amino acid or amino acid sequence adjacent to the GM-CSF protein allows splitting off of the GM-CSF protein, or else denotes a bridge member which is composed of one or more genetically codable amino acids and allows the splitting.
3. A fusion protein of the general formula Ia or Ib Met-X-Y-Z Met-Z-Y-X
(Ia) (Ib) wherein X represents the amino acid sequence of approximately the first 100 amino acids of IL-2, Z is a sequence of genetically codable amino acids representing a GM-CSF protein derivative of the formula Pro-(As)E-CSF(12-126)-K
in which (As)E denotes all or some of the first 11 amino acids of the natural GM-CSF sequence, and K denotes Glu or Asp, and Y represents a direct bond in the case where the amino acid or amino acid sequence adjacent to the GM-CSF
protein allows splitting off of the GM-CSF protein, or else denotes a bridge member which is composed of one or more genetically codable amino acids and allows the splitting.
(Ia) (Ib) wherein X represents the amino acid sequence of approximately the first 100 amino acids of IL-2, Z is a sequence of genetically codable amino acids representing a GM-CSF protein derivative of the formula Pro-(As)E-CSF(12-126)-K
in which (As)E denotes all or some of the first 11 amino acids of the natural GM-CSF sequence, and K denotes Glu or Asp, and Y represents a direct bond in the case where the amino acid or amino acid sequence adjacent to the GM-CSF
protein allows splitting off of the GM-CSF protein, or else denotes a bridge member which is composed of one or more genetically codable amino acids and allows the splitting.
4. The fusion protein as claimed in claim 1, 2 or 3, which contains at the N-terminal adjacent to the GM-CSF protein the amino acid sequence (Glu)m-(Asp)n-Pro in which m is zero or 1, and n is 1, 2 or 3.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3545568.3 | 1985-12-21 | ||
| DE19853545568 DE3545568A1 (en) | 1985-12-21 | 1985-12-21 | GM-CSF-PROTEIN, ITS DERIVATIVES, PRODUCTION OF SUCH PROTEINS AND THEIR USE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1341197C true CA1341197C (en) | 2001-03-06 |
Family
ID=6289242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000525856A Expired - Fee Related CA1341197C (en) | 1985-12-21 | 1986-12-19 | Gm-csf protein, its derivatives, the preparation of proteins of this type, and their use |
Country Status (17)
| Country | Link |
|---|---|
| EP (1) | EP0228018B1 (en) |
| JP (1) | JP2575367B2 (en) |
| KR (1) | KR940005585B1 (en) |
| AT (1) | ATE71144T1 (en) |
| AU (2) | AU601959B2 (en) |
| CA (1) | CA1341197C (en) |
| DE (2) | DE3545568A1 (en) |
| DK (1) | DK170346B1 (en) |
| ES (1) | ES2055686T3 (en) |
| FI (1) | FI91168C (en) |
| GR (1) | GR3003999T3 (en) |
| HU (1) | HU202584B (en) |
| IE (1) | IE59779B1 (en) |
| IL (1) | IL81020A (en) |
| NO (1) | NO177270C (en) |
| PT (1) | PT83972B (en) |
| ZA (1) | ZA869557B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9132146B2 (en) | 2009-12-23 | 2015-09-15 | Gradalis, Inc. | Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine |
| US9695422B2 (en) | 2009-12-23 | 2017-07-04 | Gradalis, Inc. | Furin-knockdown bi-functional RNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5078996A (en) * | 1985-08-16 | 1992-01-07 | Immunex Corporation | Activation of macrophage tumoricidal activity by granulocyte-macrophage colony stimulating factor |
| WO1987002060A1 (en) * | 1985-10-03 | 1987-04-09 | Biogen N.V. | Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells |
| DE3712985A1 (en) * | 1987-04-16 | 1988-11-03 | Hoechst Ag | BIFUNCTIONAL PROTEINS |
| US5359035A (en) * | 1985-12-21 | 1994-10-25 | Hoechst Aktiengesellschaft | Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF) |
| JP2583770B2 (en) * | 1986-09-17 | 1997-02-19 | 大塚製薬株式会社 | gene |
| JPS6420097A (en) * | 1987-03-02 | 1989-01-24 | Sumitomo Chemical Co | Human granulocyte-macrophage colony stimulating factor |
| AU609128B2 (en) * | 1987-04-02 | 1991-04-26 | Amrad Operations Pty. Limited | Leukaemia-inhibitory factor |
| PT87133B (en) * | 1987-04-02 | 1992-07-31 | Amrad Corp Ltd | METHOD OF PURIFICATION OF THE LEUKEMIA INHIBITOR FACTOR (LIF) AND PHARMACEUTICAL COMPOSITIONS CONTAINING POLIPEPTIDES WITH LIF ACTIVITY |
| WO1988010310A1 (en) * | 1987-06-25 | 1988-12-29 | Immunex Corporation | Bovine granulocyte-macrophage colony stimulating factor |
| EP0386109A1 (en) * | 1987-11-12 | 1990-09-12 | Schering Corporation | Acceleration of bone formation with gm-csf |
| GB2212159B (en) * | 1987-11-13 | 1992-01-22 | British Bio Technology | Synthetic gene for human granulocyte/macrophage colony stimulating factor. |
| EP0413721A4 (en) * | 1988-04-21 | 1991-11-13 | Medvet Science Pty. Ltd. | Human gm-csf variants |
| WO1990012877A1 (en) * | 1989-04-19 | 1990-11-01 | Cetus Corporation | Multifunctional m-csf proteins and genes encoding therefor |
| WO1991002754A1 (en) * | 1989-08-22 | 1991-03-07 | Immunex Corporation | Fusion proteins comprising gm-csf and il-3 |
| NZ236819A (en) * | 1990-02-03 | 1993-07-27 | Max Planck Gesellschaft | Enzymatic cleavage of fusion proteins; fusion proteins; recombinant dna and pharmaceutical compositions |
| US5270181A (en) * | 1991-02-06 | 1993-12-14 | Genetics Institute, Inc. | Peptide and protein fusions to thioredoxin and thioredoxin-like molecules |
| US6060258A (en) * | 1995-09-28 | 2000-05-09 | Otsuka Pharmaceutical Co., Ltd. | Neutrophil chemotactic lymphokine, and method for the diagnosis of drug hypersensitive granulocytopenia using the same |
| KR100448021B1 (en) * | 2001-12-28 | 2004-09-08 | 크레아젠 주식회사 | Transgenic Escherichia coli transformed with pGM-CSF vector expressing recombinant mouse granulocyte-macrophage colony stimulating factor and method for mass production of the recombinant protein |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4438032A (en) * | 1981-01-30 | 1984-03-20 | The Regents Of The University Of California | Unique T-lymphocyte line and products derived therefrom |
| SE8300693L (en) * | 1983-02-09 | 1984-08-10 | Sven Lofdahl | SET TO MAKE AND ISOLATE PROTEINS AND POLYPEPTIDES, AND A HYBRID VECTOR FOR THIS |
| IL71991A (en) * | 1983-06-06 | 1994-05-30 | Genentech Inc | Preparation of mature human IGF and EGF via prokaryotic recombinant DNA technology |
| AU594014B2 (en) * | 1984-03-21 | 1990-03-01 | Research Corporation Technologies, Inc. | Recombinant DNA molecules |
| EP0188479B1 (en) * | 1984-07-06 | 1991-09-11 | Sandoz Ag | Lymphokine production and purification |
| ZA856108B (en) * | 1984-10-29 | 1986-10-29 | Immunex Corp | Cloning of human granulocyte-macrophage colony simulating factor gene |
| AU588819B2 (en) * | 1984-10-29 | 1989-09-28 | Immunex Corporation | Cloning of human granulocyte-macrophage colony stimulating factor gene |
| KR920003822B1 (en) * | 1984-11-20 | 1992-05-15 | 쉐링 바이오텍 코포레이션 | Polypeptides exhibiting human granulocyte macrophage and eosinophil cellular growth factor activity |
| WO1987002060A1 (en) * | 1985-10-03 | 1987-04-09 | Biogen N.V. | Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells |
| DE3541856A1 (en) * | 1985-11-27 | 1987-06-04 | Hoechst Ag | EUKARYOTIC FUSION PROTEINS, THEIR PRODUCTION AND USE, AND MEANS FOR CARRYING OUT THE PROCESS |
| DE3636903A1 (en) * | 1985-12-21 | 1987-07-02 | Hoechst Ag | FUSION PROTEINS WITH EUKARYOTIC BALLASTES |
-
1985
- 1985-12-21 DE DE19853545568 patent/DE3545568A1/en not_active Withdrawn
-
1986
- 1986-12-16 DE DE8686117484T patent/DE3683267D1/en not_active Expired - Lifetime
- 1986-12-16 AT AT86117484T patent/ATE71144T1/en not_active IP Right Cessation
- 1986-12-16 EP EP86117484A patent/EP0228018B1/en not_active Expired - Lifetime
- 1986-12-16 ES ES86117484T patent/ES2055686T3/en not_active Expired - Lifetime
- 1986-12-18 FI FI865186A patent/FI91168C/en not_active IP Right Cessation
- 1986-12-18 IL IL8102086A patent/IL81020A/en active IP Right Grant
- 1986-12-19 CA CA000525856A patent/CA1341197C/en not_active Expired - Fee Related
- 1986-12-19 NO NO865191A patent/NO177270C/en unknown
- 1986-12-19 PT PT83972A patent/PT83972B/en not_active IP Right Cessation
- 1986-12-19 DK DK619086A patent/DK170346B1/en not_active IP Right Cessation
- 1986-12-19 ZA ZA869557A patent/ZA869557B/en unknown
- 1986-12-19 HU HU865355A patent/HU202584B/en not_active IP Right Cessation
- 1986-12-19 AU AU66756/86A patent/AU601959B2/en not_active Ceased
- 1986-12-19 IE IE333586A patent/IE59779B1/en not_active IP Right Cessation
- 1986-12-20 KR KR1019860011011A patent/KR940005585B1/en not_active IP Right Cessation
- 1986-12-22 JP JP61306186A patent/JP2575367B2/en not_active Expired - Lifetime
-
1990
- 1990-09-19 AU AU62683/90A patent/AU637139B2/en not_active Ceased
-
1992
- 1992-03-11 GR GR920400222T patent/GR3003999T3/el unknown
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9132146B2 (en) | 2009-12-23 | 2015-09-15 | Gradalis, Inc. | Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine |
| US9695422B2 (en) | 2009-12-23 | 2017-07-04 | Gradalis, Inc. | Furin-knockdown bi-functional RNA |
| US9790518B2 (en) | 2009-12-23 | 2017-10-17 | Gradalis, Inc. | Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine |
| US10253331B2 (en) | 2009-12-23 | 2019-04-09 | Gradalis, Inc. | Furin-knockdown and GM-CSF-augmented (FANG) cancer vaccine |
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