CA1339894C - Fusion proteins with a eukaryotic ballast portion - Google Patents
Fusion proteins with a eukaryotic ballast portionInfo
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- CA1339894C CA1339894C CA000525858A CA525858A CA1339894C CA 1339894 C CA1339894 C CA 1339894C CA 000525858 A CA000525858 A CA 000525858A CA 525858 A CA525858 A CA 525858A CA 1339894 C CA1339894 C CA 1339894C
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/55—IL-2
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
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- C07K2319/00—Fusion polypeptide
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- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
- C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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Abstract
A suitable "ballast portion" for fusion proteins is part of the amino acid sequence of interleukin-2 (IL-2) which contains significantly fewer than 100 amino acids. It is advantageous to start from a synthetic IL-2 gene which is divided by unique cleavage sites into six segments, up to three of which can be linked in arbitrary sequence by the modular principle. The invention permits specific constructions with which the solubility of the fusion protein can be altered.
Description
13'~983~
HOECHST AKTIENGESELLSCHAFT HOE 86/F271J Dr.KL/mu Description Fusion proteins with a eukaryotic balla~t portion Fusion proteins having a C- or N-terminal portion essen-tially corresponding to the first 100 amino acids of inter-leukin-2 have already been proposed (German Patent Appli-cation P 3,541,856.7). The interleukin-2 portion in these may be derived from mammalian interleukin-2, for example from mouse or rat interleukin-2, which are discLosed in European Patent Application with the publication number (hereinafter "EP-A") 0,091,539, but preferably from human interleukin-2. These fusion proteins are surprisingly stable in the host cell and can, by reason of their low solubility, easily be separated from the soluble proteins intrinsic to the host.
In a further development of this inventive concept, it has now been found, surprisingly, that even considerably smaller portions of the interleukin-2 molecule are suit-able as "ballast" portion for fusion proteins of this type. The invention is defined in the patent claims.
Preferred embodiments are explained in detail hereinafter.
It is particularly advantageous to start from the syn-thetic gene for human interleukin-2 (hereinafter "IL-2") which is described in EP-A 0,163,249 and depicted in the addendum. This synthetic gene contains a number of unique restriction cleavage sites which permit the DNA coding for IL-2 to be broken do~n into "manageable" segments. Using these segments it is possible by the modular principle to tailor the ballast portion for fusion proteins, the solu-bility of the fusion proteins obtained ranging from high to low depending on the combination of the segments and depending on the nature of the desired protein.
Thus the invention allows the solubility to be directed to~ards that which is most advantageous for the possible ~ or desired working up of the product, that is to say high solubility when the product is to be purified by chroma-tography, for example using an antibody column, or low solubility if, for pre-purification, the soluble proteins intrinsic to the host are to be removed, for example by centrifugation.
A particular advantage of the invention is that it is possible to prepare fusion proteins having a very small "ballast portion", since this results in the relative - yield of desired protein being considerably increased.
Another advantage of the invention is that the '!baLlast portion" can be constructed in such a way that it impairs the spatial structure of the desired protein as little as possible and thus, for example, does not prevent folding up .
Cleavage of the fusion proteins results in not only the desired protein but also the "ballast portion", that is to say the IL-2 derivative. This may have IL-2 activity (T-cell proliferation test) or bind to IL-2 receptors.
The "modular principle" according to the invention can thus also be used to produce, as "by-products" IL-2 deriva-tives which have the biological activities of IL-2 to a greater or lesser extent.
Particularly advantageous embodiments of the invention are explained hereinafter with reference to the synthetic gene described in EP-A 0,163,249. This gene is cut at the 5' end with the restriction endonuclease EcoRI and at the 3' end with SalI. Apart from the three unique restriction cleavage sites for the enzymes PstI, XbaI and SacI, which were used to construct this gene, the locations of the unique cleavage sites for MluI and PvuI are also favorable.
When the sequences located between these cleavage sites are designated A to F, the synthetic gene can be repre-sented diagrammatically as follows (EcoRI)-A-PstI-B-MluI-C-XbaI-D-SacI-E-PvuI-F-(SalI) The segments A to F are thus particularly suitable "units"
for the modular system according to the invention. Thus, in this representation the "ballast portion" for the fu-sion proteins described in German Patent Application P 3,541,856.7 corresponds to the segments A to E, and that for the bifunctional protein having the entire IL-2 gene, ~hich is mentioned in the same application, corre-sponds to all the segments A to F. In contrast, the geneconstructs according to the invention relate to other combinations of the segments A to F, preferably having fe~er than 4 of these segments, the segment A coding for the N-terminal end of the fusion protein. ~he arrangement of the other segments is arbitrary, use optionally being made of appropriate adaptors or linkers. Appropriate adaptor or linker sequences can also be introduced at the C-terminal end of the "ballast portion", and in this case they can code for amino acids or short amino acid sequences ~hich permit or facilitate the cleavage off, enzymatically or chemically, of the "ballast portion" from the desired protein. The adaptor or linker sequences can, of course, also be used to tailor the "ballast portion" for a parti-cular fusion protein, for example to achieve a desired solubility. In this context, it has emerged, surprisingly, that the solubility of the fusion proteins does not depend on the molecular size but that, on the contrary, even rel-atively small molecules may have lo~ solubility. Thus, with knowledge of these relationships, ~hich are explained in detail in the examples, those skilled in the art are able ~ithout great experimental effort to obtain fusion proteins according to the invention ~ith a small "ballast portion" and having particular desired properties.
Thus, if the desired protein is a eukaryotic protein the fusion proteins obtained according to the invention are composed exclusively or virtually exclusively of eukaryo-tic protein sequences. However, surprisingly, these fusion proteins are not recognized as foreign proteins by 133989~
-~ 4 - the prokaryotic host cells, and are not rapidly degraded by proteases intrinsic to the host. This degradation takes place particularly often in the case of proteins which are foreign to the host and coded for by cDNA sequences which are to be expressed in bacteria. It has now emerged that cDNA sequences can be expressed very effectively if they are "embedded" in the segments according to the invention.
It is possible to construct specific vectors for this pur-pose, which contain between the sequences according to the invention a polylinker sequence which has several cloning sites for the cDNA sequences. ~here the cDNA which has been cloned in contains no stop codon the polypeptide sequence coded for by the cDNA sequence is additionally protected by the polypeptide for which the C-terminal segment codes.
The fusion proteins can be cleaved chemically or enzymati-cally in a manner known per se. The choice of the suit-able method depends, in particular, on the amino acid sequence of the desired protein. If the latter contains, for example, no methionine it is possible for the connect-ing element to denote Met, in-which case chemical cleavage with cyanogen chloride or bromide is carried out. If there is a cysteine at the carboxyl terminal end of the connect-ing element, or if the connecting element represents Cys,then it is possible to carry out a cysteine-specific enzy-matic cleavage or chemical cleavage, for example after specific S-cyanylation. If there is a tryptophan at the carboxyl terminal end of the bridging element, or if the connecting element represents Trp, then chemical cleavage with N-bromosuccinimide can be carried out.
Desired proteins which do not contain Asp - Pro in their amino acid sequence and are sufficiently stable to acid can, as fusion proteins with this bridging element, be cleaved proteolytically in a manner known per se. This results in proteins which contain N-terminal proline or C-terminal aspartic acid. It is therefore also possible in this way to synthesize modified proteins.
1339~9~
The Asp-Pro bond can be made even more labile to acid if - this bridging element is (Asp)n-Pro or Glu-(Asp)n-Pro, n denoting 1-to 3.
Examples of enzymatic cleavages are likewise known, it also being possible to use modified enzymes of improved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, then the chosen sequence is advantageously a peptide sequence in which an amino acid which can be split off with trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of proinsulin, for example Ala-Ser-Met-Thr-Arg, since in this case the arginine-specific cleavage can be carried out with the protease trypsin.
If the desired protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, then the fusion protein with the appropriate bridging element can be cleaved with factor Xa (EP-A 0,025,190 and 0,161,973).
The fusion protein is obtained by expression in a suit-able expression system in a manner known per se. All known host-vector systems are suitable for this purpose, that is to say, for example, mammalian cells and micro-organisms, for example yeasts and, preferably, bacteria, in particular E. coli.
The DNA sequence which codes for the desired protein is incorporated in a known manner into a vector ~hich ensures satisfactory expression in the selected expression system.
In bacterial hosts, it is advantageous to select the pro-moter and operator from the group comprising lac, tac, trp, PL or PR of phage ~, hsp, omp or a synthetic pro-moter as proposed in, for example, German Offenlegungs-schrift 3,430,683 (EP-A 0,173,149). The tac promoter-operator-sequence is advantageous and is now commercially - avai.able (for example expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equip-ment for Molecular Biology", 1984, page 63).
S In the expression of the fusion protein according to the invention it may prove advantageous to modify individual tripLets for the first few amino acids downstream of the ATG start codon in order to prevent any base-pairing at the mRNA level. Modifications of this type, as well as modifications, deletions or additions of individual amino acids in the IL-2 protein portion, are familiar to those skilled in the art, and the invention likewise relates to them. Elimination of cysteine or replaçement of cysteine by other amino acids, in order to prevent formation of undesired disulfide bridges, as is disclosed in, for ex-ample, EP-A 109,748, may be mentioned by way of example.
Figures 1 to 13 illustrate in the manner of a flow diagram the processes of the syntheses described in the examples having the same numbers. To facilitate comprehension, the preparation of the starting materials and intermediates has been depicted in Figures A to C. For the sake of clarity the reference numbers in Figures 1 to 13 each start a new decade, thus (11) in Figure 1. Reference numbers of starting materials to which the present appli-cation does not relate end with zero, thus, for example, (20) in Figure 2. The figures are not drawn to scale, in particular the scale is expanded appropriately in the region of the polylinker sequences. IL-2 sequences are defined by thick lines, and structural genes for desired proteins are emphasized in other ways.
Figure A gives an overview of the segments A to F accord-ing to the invention and of the combination of segments A and B. The starting material is the plasmid p159/6, whose preparation is described in detail in EP-A 0,163,249 and which is defined by Figure 5 in this publication.
Figure 3 shows the expression plasmid pE~1000, whose - 7 _ 13398~4 preparation is described in German Patent Application P 3,541,856.7 and is shown in Figure 1 therein. This plasmid is opened in the polylinker sequence by appropri-ate double digestion, this resulting in the linearized S plasmids (Ex1) to (Ex4).
Figure C shows the preparation of the pUC12 derivative p~226 and of the expression plasmid p~Z26-1, both of which contain segments A and F separated by a polylinker se-quence.
Figure 1 shows the preparation of the pUC12 derivativepKH40 and of the expression plasmid pK40, which code for fusion proteins in which the protein sequence correspond-ing to segment A, that is to say the first 22 amino acidsof IL-2, is followed by the bridging element Thr-Arg, with subsequently the amino acid sequence of proinsulin.
Figure 2 shows the construction of the plasmid pSL11 and of the expression plasmid pSL12, which code for polypep-tides in which the segment A is followed by a bridging element corresponding to polylinker sequences t2) and (20a), with subsequently the amino acid sequence of proinsulin.
Figure 3 shows the construction of the expression plasmid pK50 which codes for a polypeptide in which segments A
and B, that is to say the first 38 amino acids of IL-Z, are dlrectly followed by the amino acid sequence of pro-insul ln.
30Figure 4 shows the construction of the expression plasmid pKS1 which codes for a polypeptide in which segments A
and B are followed by a bridging element corresponding to sequences (42) and (41), to which is connected the amino acid sequence of proinsulin.
Figure 5 shows the construction of the expression plasmid pK52 which differs from pKS1 by the inserted MluI linker (51) which codes for the amino acid sequence which permits - - 8 - 1~3~9~
cleavage with factor Xa. pKS2 can also be obtained from pKS0 (Figure 3) by cleavage with MluI and introduction of the said MluI linker.
S Figure 6 shows the construction of the expression plasmid pK53 from pKS1 (Figure 4), likewise by introduction of the MluI linker.
Figure 7 shows the construction of the expression plasmid pSL14 from pSL12 (Figure 2) by introduction of the frag-ment C into the polylinker. This results in direct attach-ment of the segment C to the segment A. In the following polylinker the first two amino acids (each Glu) corre-spond to amino acids 60 and 61 of IL-2. Thus the IL-2 portion is composed of amino acids 1 to 22 and 37 to 61.
The subsequent amino acid sequence corresponds to that which is coded for by the plasmid pSL12 (Figure 2).
Figure 8 shows the construction of the expression plasmid pPH31 which codes for a fusion protein in which segments A to C are followed by a bridging element which is re-presented by sequence (81), with subsequently the amino acid sequence of proinsulin.
Figure 9 shows the construction of the plasmid pK192 which codes for a fusion protein in which segements A and B are followed by methionine and, thereafter, the amino acid sequence of hirudin.
Figure 10 shows the construction of the plasmid pW214 which codes for a fusion protein in which segments A and B are followed by the amino acid sequence which permits cleavage with factor Xa, with subsequently the amino acid sequence of granulocyte/macrophage colony stimulating factor (CSF).
Figure 11 shows the construction of the expression plasmid p~233 which codes for a fusion protein in which segments A and C (corresponding to amino acids 1 to 22 and 37 to 61 of IL-2) are followed by the bridging element Leu-Thr-Ile-Asp-Asp-Pro, with subsequently the amino acid sequence of CSF.
Figure 12 shows the construction of the expression plasmid p~234 which codes for a fusion protein having the follow-ing amino acid sequence: Segment A (amino acids 1 to 22) is followed by a bridging element Thr-Arg, then by segment D (amino acids 59 to 96 of IL-2), by Thr-Asp-Asp-Pro as a further connecting element, and finally by CSF.
Figure 13 shows the construction of the plasmids pH200 and pH201 and of the expression plasmid pH202. These plasmids have a polylinker located between segments A and F or A, P and F, into whose numerous cleavage sites foreign DNA can be cloned. These plasmids are particularly suitable for cloning cDNA sequences.
The invention is explained in detail in the examples which follow, in which the numbering coincides with that in the figures. Unless otherwise stated, percentage data relate to weight.
Example A
The starting plasmid p159/6 is described in EP-A 0,163,249 (Figure S). The sequence defined there as "IL-2" or in the text as "DNA sequence I" is in Figure A divided into segments A to F which are bounded by cleavage sites for the enzymes EcoRI, PstI, MluI, XbaI, SacI, PvuI and SalI.
Double digestion with the appropriate enzymes results in the segments (A) to (F) or adjoined segments, for example the segment (A,B) with EcoRI and MluI.
Example E
The preparation of the expression plasmid pEW1000 has been proposed in the (not prior-published) German Patent Appli-cation P3,541,856.7 (Figure 1). This plasmid is a derivative of the plasmid ptac11 (Amann et al., Gene 25 (1983) 167 - 178) into whose recognition site for EcoRI
has been incorporated a synthetic sequence which contains a SalI cleavage site. In this way the expression plasmid S pKK177.3 is obtained. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 - 769) results in the plasmid pJF118. This is opened at the unique restriction cleavage site for AvaI, and is, in a known manner, short-ened by about 1000 bp by exonuclease treatment and is ligated. This results in the plasmid pEW1000. Opening this plasmid in the polylinker using the enzymes EcoRI
and HindIII, SalI, PstI or SmaI results in the linearized expression plasmids (Ex1), (Ex2), (Ex3) and (Ex4).
Example C
The commercially available plasmid pUC12 is opened with EcoRI and SalI, and the linearized plasmid (1) is iso-lated. Ligation of (1) with the segment (A), the synthe-tic linker sequence (2) and the segment (F) results inthe plasmid p~226 (3).
The strain E. coli 79/02 is transformed in a known manner with the plasmid DNA from the ligation mixture. The cells are plated out on agar plates which contain isopropyl-B-D-thiogalactopyranoside (IPTG), S-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal) and 20 ~g/ml ampicillin (Ap).
The plasmid DNA is obtained from white clones, and the formation of the plasmid (3) is confirmed by restriction analysis and DNA sequence analysis.
The small EcoRI-HindIII fragment (4) is cut out of the plasmid (3) and is isolated. This fragment is ligated with the linearized expression plasmid (Ex1) in a T4 DNA
ligase reaction. The resulting plasmid pW226-1 (S) is characterized by restriction analysis.
Competent cells of the strain E. coli Mc 1061 are trans-formed with DNA from the plasmid pW 226-1. Clones which 133989~
are resistant to ampicillin are isolated on Ap-containing agar plates. The plasmid DNA is reisolated from Mc 1061 cells and then characterized anew by restriction analysis.
Competent cells of the E. coli strain W 3110 are now transformed with plasmid DNA isolated from E. coli Mc 1061 cells. E. coli ~ 3110 cells are always used for expression hereinafter. All the expression experiments in the stated examples are carried out in accordance with the following conditions.
An overnight culture of E. coli cells which contain the plasmid (5) is diluted in the ratio of about 1:100 with LB medium (J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) which contains 50 ~g/ml ampicillin, and growth is followed by measurement of the OD. ~hen the OD is 0.5 the culture is adjusted to 1 mM in IPTG and, after 150 to 180 minutes, the bacteria are spun down. The bacteria are boiled in a buffer mix-ture (7M urea, 0.1% SDS, 0.1 M sodium phosphate, pH 7.0) for 5 minutes, and samples are applied to a SDS gel electrophoresis plate. After electrophoresis, bacteria which contain the plasmid (5) produce a protein band which corresponds to the size of the expected protein (6 kD).
The stated induction conditions apply to shake cultures;
for larger fermentations appropriate modifications of the OD values and, where appropriate, slight variations in the IPTG concentrations are advantageous.
The resulting protein shows no biological activity in a cell proliferation test with an IL-2-dependent cell line (CTLL 2).
Example 1 The plasmid (3) is opened with MluI and SalI, and the two resulting fragments are separated by gel electrophoresis.
The larger fragment (11) is isolated.
- 12 - 1 ~ 3 9 ~ 9 4 The synthetic oligonucleotide (12) is ligated with the blunt-ended DNA (13) coding for proinsulin (Wetekam et al., ~ene 19 (1982) 179 - 183), this resulting in DNA sequence (14). The latter is cut with MluI and SalI, this result-ing in DNA sequence (15). The latter is now ligated witht~7e fragment (11), this resulting in formation of the pLasmid pKH40 (16). The latter is characterized by re-striction analysis.
The plasmid (16) is digested with EcoRI and HindIII, and the small fragment (17) is isolated by gel electrophoresis.
Ligation with the linearized expression plasmid (Ex1) re-sults in the expression plasmid pK40 (18). Expression as indicated in Example C results in a protein which, after cell disruption, is found in the soluble fraction of cellular protein. The Western blot technique is used to demonstrate that the proinsulin sequence is intact.
Example 2 The starting material is the plasmid pPH30 which is de-picted in the (not prior-published) German Patent Appli-cation P 3,541,856.7, in Figure 3c. Within the meaning of the present invention, in Figure 2 the IL-2 part-sequence is shown as "A-E" (20). The end of this se-quence and the bridging element up to the proinsulin se-quence is shown as (20a) in Figure 2.
The plasmid (20) is digested with PvuI and HindIII, and the smalL fragment (22) is isolated. In addition, the plasmid (3) is opened with EcoRI and PvuI, and the small fragment (23) is isolated. Moreover, the vector pUC12 is digested with EcoRI and HindIII, and the large fragment (21) is isolated. Ligation of the fragments (21), (23) and (22) results in the plasmid pSL11 (24).
The plasmid (24) is digested with HindIII and partially with EcoRI, and the fragment (25) which contains the segment A and the proinsulin gene is isolated. Ligation of (25) into the linearized expression plasmid (Ex1) re-sults in the expression plasmid pSL12 (26).
Expression as indicated in Example C and subsequent work-ing up results in a soluble fusion protein. ~estern blot analysis with insulin antibodies confirms that this pro-tein contains the intact insulin sequence.
Example 3 The plasmid ptrpED5-1 (30) (Hallewell et al., Gene 9 (1980) 27-47) is used for amplification of the proinsulin gene.
The plasmid is opened with HindIII and SalI, and the large fragment (31) is isolated. The fragment (31) is ligated with DNA sequence (14), this resulting in the plasmid pH106/4 (32).
The plasmid (32) is digested with SalI and MluI, and the small fragment (15) is isolated. The linearized expres-sion plasmid (Ex2), the segment (A,B) and the fragment(15) are now ligated, this resulting in the expression plasmid pK50 (33).
Expression of the coded fusion protein is carried out as indicated in Example C. The cells are then spun down from the culture broth and ruptured in a French press.
The protein suspension is now centrifuged to separate it into its soluble and insoluble protein constituents. The two fractions are analyzed by gel electrophoresis in a known manner on 17.5% SDS polyacrylamide gels and subse-quently by staining the proteins with the dyestuff Coo-massie blue. It is found, surprisingly, that the fusion protein is located in the insoluble sediment. Western blot analysis with insulin antibodies confirms that in-tact proinsulin is present in the fusion protein.
The sediment from the French press disruption can now im-mediately be used further for isolation of proinsulin.
- - 14 - I 3 3 9 8 9 ~
Example 4 The starting material is the plasmid pPH20 (40) which is depicted in German Patent Application P 3,541,856.7, in S Figure 3c. Cutting this plasmid with EcoRI, filling in the protruding ends and cutting with HindIII results in the fragment (41), from which the DNA sequence of the part of (40) which is of interest here can be seen.
Ligation of the linearized expression plasmid (Ex4) with the segment (A,B) the synthetic oligonucleotide (42) and the fragment (41) results in the plasmid pK51 (43).
Example 5 Ligation of the linearized expression plasmid (Ex2) with the segment (A,B), the synthetic oligonucleotide (51) and the DNA sequence (15) results in the plasmid pK52 (52).
The correct orientation of the oligonucleotide (51) is established by sequence analysis. The plasmid codes for a fusion protein which contains the amino acid sequence which corresponds to the oligonucleotide (51) and thus can be cleaved by activated Factor Xa.
The plasmid (52) can also be obtained in the following manner:
Partial cutting of the plasmid (33) with MluI and ligation of the resulting opened plasmid (53) with the DNA sequence (51) likewise results in the plasmid pK52.
Example 6 Partial cutting of the plasmid (43) with MluI and ligation of the resulting linearized plasmid (61) with the synthe-tic DNA sequence (51) results in the plasmid pK53 (62).The latter likewise codes for a fusion protein which can be cleaved with activated factor Xa. The correct orient-ation of the sequence (51) is established, as in Example 5, by DNA sequence analysis.
Example 7 The plasmid (26) is cleaved with XbaI and partially with MluI, and the large fragment (71) is isolated. Ligation with the segment (C) results in the plasmid pSL14 (72).
After expression and cell disruption, the fusion protein is found in the soluble fraction of cellular protein.
Example 8 The plasmid (20) is cleaved with XbaI and partially with EcoRI, and the protruding ends are fiLled in, this result-ing in DNA sequence (81). Ligation under blunt end con-ditions results in the plasmid pPH31 (82). The fusion protein is found in the insoluble fraction of cellular protein.
Example 9 The starting material used is the plasmid (90) which is described in EP-A 0,171,024 (Figure 3). This plasmid is - reacted with SalI and then with AccI, and the small frag-ment (91) is isolated. The latter is ligated with the synthetic oligonucleotide (92), this resulting in DNA
sequence (93). The latter is cut with MluI, this result-ing in DNA fragment (94).
The plasmid (33) is digested with MluI, partially, and ~ith SalI, and the large fragment (95) is isolated. The latter is ligated with the DNA sequence (94), this result-ing in the expression plasmid pK192 (96). The latter codes for a fusion protein in which the first 38 amino acids of IL-2 are followed by methionine and then by the amino acid sequence of hirudin. The fusion protein is found in the soluble fraction of cellular protein.
Example 10 The starting material used is the plasmid pHG23 (100) - 16 - I~ 3 9 8 9 ~
which is described in EP-A 0,183,350 and which is gener-ally accessible from the American Type Culture Collection under No. ATCC 39000. This plasmid is cut with SfaNI, the protruding ends are filled in, then reaction with PstI is carried out, and the small fragment (101) is iso-lated. Ligation of the linearized expression plasmid (Ex3) with the segment (A,~), the synthetic oligonucleo-tide (102) and the fragment (101) results in the express-ion plasmid p~214 (103). This plasmid codes for a fusion protein in which the first 38 amino acids of IL-2 are followed by the sequence which is derived from the oligo-nucleotide (102) and which allows the molecule to be cleaved with factor Xa, with subsequently the am;no acid sequence of CSF. After cell disruption, the fusion pro-tein is found in the insoluble fraction of cellular pro-tein.
Example 11 The starting plasmid p~216 (110) is proposed in German Patent Application P 3,545,568.3 (Figure 2b). In this plasmid, the IL-2 sequence corresponding to segments A
to E (PvuI cleavage site) is followed by a linker which codes for the amino acids Asp-Asp-Pro, immediately followed by the amino acid sequence for CSF. The connecting se-quence between IL-2 and CSF allows the fusion protein to be cleaved proteolytically.
The sequence (111) is isolated from the plasmid (110) by cutting with PvuI and HindIII.
The plasmid t3) is cut with MluI and XbaI, and the large fragment (112) is isolated. The latter is ligated with the segment (C), this resulting in the plasmid pW227 (113).
This plasmid is reacted with EcoRI and HindIII, and the short fragment (114) is isolated. If this fragment is ligated with the linearized expression plasmid (Ex1) the result is the plasmid p~227-1 (115). The plasmid codes for a protein which is derived from IL-2 but which has no 133989~
IL-2 activity.
The plasmid (113) is additionally cut ~ith EcoRI and Pvu~, and the short fragment (116) is isolated. Ligation of the linearized expression plasmid (Ex1) with the fragments (116) and (111) results in the expression plasmid pW233 (117). The latter codes for an insoluble fusion protein ~hich, by reason of the abovementioned linker, can be cleaved proteolytically.
Example 12 The plasmid (3) is cut with XbaI and SacI, and the large fragment (121) is isolated. Ligation with the segment (D) results in the plasmid pW228 (122). The latter is cut ~ith EcoRI and HindIII, and the small fragment (123) is isolated. Ligation of the linearized expression plas-mid (Ex1) ~ith the fragment (123) results in the express-ion plasmid pW228-1 (124). This plasmid codes for a bio-logically inactive IL-2 derivative. The plasmid is diges-ted ~ith EcoRI and PvuI, and the short fragment (125) is isolated. Ligation of the linearized expression plasmid (Ex1) with the fragments (125) and (111) results in the expression plasmid p~234 (126). The latter codes for a sparingly soluble fusion protein which can likewise be cleaved proteolytically.
Example 13 For the construction of plasmids which are suitable, in particular, for the expression of cDNA sequences, initially the polylinker sequence (131) is synthesized.
Ligation of the linearized plasmid (1) ~ith the segment (A), the polylinker sequence (131) and segment (F) results in the plasmid pH200 (132).
The plasmid (132) is reacted ~ith EcoRI and MluI, and the large fragment (133) is isolated. Ligation of the latter with the segment (A,B) results in the plasmid pH201 (134).
The plasmid (134) is reacted with EcoRI and HindIII, a~nd tne short fragment (135) is isolated. Ligation of this fragment with the linearized expression plasmid (Ex1) re-sults in the expression plasmid pH 202 (136).
The plasmid (136) is opened with BamHI, and the cDNA which is to be expressed is introduced into the linearized plas-mid via a commercially available BamHI adaptor. Depend-ing on the orientation of the cDNA, every third sequence is attached to (A,B) in the reading frame. If the cDNA
sequence contains no stop codon the polypeptide sequence for which it codes is additionally protected by the amino acid sequence corresponding to the segment (F).
If the cDNA is not connected in the correct reading frame, a shift of the reading frame is brought about by, for example, cleaving the cDNA-containing (original or multiplied) plasmids with MluI or XbaI (as long as the cDNA does not contain cleavage sites for these enzymes) and filling in the protruding ends by a Klenow polymerase reaction.
Addendum Triplet No. 0 1 ~ 2 Amino acid Met Ala Pro Nucleotide No. 1 10 Cod. strand 5 ' AA TTC ATG GCG CCG
Non-cod. strand 3' G TAC CGC GGC
(EcoRI) Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG
TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC
Gln Leu Glu His Leu Leu Leu Asp Leu Gln CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG
GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC
PstI
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys ATG ATC CTG AAC GGT ATC AAC AAC TAC AAA
TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC
TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG
MluI
Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA
TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT
Leu Lys His Leu Gln Cys Leu Glu Glu Glu CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG
GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC
XbaI
Leu Lys Pro Leu Glu Glu Val Leu Asn Leu CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG
GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC
Ala Gln Ser Lys Asn Phe His Leu Arg Pro GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG
CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC
GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG
Val Leu Glu Leu Lys Gly Ser Glu Thr Thr GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG
CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC
SacI
.
133989~
Phe Met Cys Glu Tyr Ala Asp Glu ~ Thr Ala TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG
AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC
TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG
PvuI
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu ACC TTC TGC CAG TCG ATC ATC TCT ACC CTG
TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC
Thr ACC TGA TAG 3 ' TG6 ACT ATC AGC T S' ( S a l I )
HOECHST AKTIENGESELLSCHAFT HOE 86/F271J Dr.KL/mu Description Fusion proteins with a eukaryotic balla~t portion Fusion proteins having a C- or N-terminal portion essen-tially corresponding to the first 100 amino acids of inter-leukin-2 have already been proposed (German Patent Appli-cation P 3,541,856.7). The interleukin-2 portion in these may be derived from mammalian interleukin-2, for example from mouse or rat interleukin-2, which are discLosed in European Patent Application with the publication number (hereinafter "EP-A") 0,091,539, but preferably from human interleukin-2. These fusion proteins are surprisingly stable in the host cell and can, by reason of their low solubility, easily be separated from the soluble proteins intrinsic to the host.
In a further development of this inventive concept, it has now been found, surprisingly, that even considerably smaller portions of the interleukin-2 molecule are suit-able as "ballast" portion for fusion proteins of this type. The invention is defined in the patent claims.
Preferred embodiments are explained in detail hereinafter.
It is particularly advantageous to start from the syn-thetic gene for human interleukin-2 (hereinafter "IL-2") which is described in EP-A 0,163,249 and depicted in the addendum. This synthetic gene contains a number of unique restriction cleavage sites which permit the DNA coding for IL-2 to be broken do~n into "manageable" segments. Using these segments it is possible by the modular principle to tailor the ballast portion for fusion proteins, the solu-bility of the fusion proteins obtained ranging from high to low depending on the combination of the segments and depending on the nature of the desired protein.
Thus the invention allows the solubility to be directed to~ards that which is most advantageous for the possible ~ or desired working up of the product, that is to say high solubility when the product is to be purified by chroma-tography, for example using an antibody column, or low solubility if, for pre-purification, the soluble proteins intrinsic to the host are to be removed, for example by centrifugation.
A particular advantage of the invention is that it is possible to prepare fusion proteins having a very small "ballast portion", since this results in the relative - yield of desired protein being considerably increased.
Another advantage of the invention is that the '!baLlast portion" can be constructed in such a way that it impairs the spatial structure of the desired protein as little as possible and thus, for example, does not prevent folding up .
Cleavage of the fusion proteins results in not only the desired protein but also the "ballast portion", that is to say the IL-2 derivative. This may have IL-2 activity (T-cell proliferation test) or bind to IL-2 receptors.
The "modular principle" according to the invention can thus also be used to produce, as "by-products" IL-2 deriva-tives which have the biological activities of IL-2 to a greater or lesser extent.
Particularly advantageous embodiments of the invention are explained hereinafter with reference to the synthetic gene described in EP-A 0,163,249. This gene is cut at the 5' end with the restriction endonuclease EcoRI and at the 3' end with SalI. Apart from the three unique restriction cleavage sites for the enzymes PstI, XbaI and SacI, which were used to construct this gene, the locations of the unique cleavage sites for MluI and PvuI are also favorable.
When the sequences located between these cleavage sites are designated A to F, the synthetic gene can be repre-sented diagrammatically as follows (EcoRI)-A-PstI-B-MluI-C-XbaI-D-SacI-E-PvuI-F-(SalI) The segments A to F are thus particularly suitable "units"
for the modular system according to the invention. Thus, in this representation the "ballast portion" for the fu-sion proteins described in German Patent Application P 3,541,856.7 corresponds to the segments A to E, and that for the bifunctional protein having the entire IL-2 gene, ~hich is mentioned in the same application, corre-sponds to all the segments A to F. In contrast, the geneconstructs according to the invention relate to other combinations of the segments A to F, preferably having fe~er than 4 of these segments, the segment A coding for the N-terminal end of the fusion protein. ~he arrangement of the other segments is arbitrary, use optionally being made of appropriate adaptors or linkers. Appropriate adaptor or linker sequences can also be introduced at the C-terminal end of the "ballast portion", and in this case they can code for amino acids or short amino acid sequences ~hich permit or facilitate the cleavage off, enzymatically or chemically, of the "ballast portion" from the desired protein. The adaptor or linker sequences can, of course, also be used to tailor the "ballast portion" for a parti-cular fusion protein, for example to achieve a desired solubility. In this context, it has emerged, surprisingly, that the solubility of the fusion proteins does not depend on the molecular size but that, on the contrary, even rel-atively small molecules may have lo~ solubility. Thus, with knowledge of these relationships, ~hich are explained in detail in the examples, those skilled in the art are able ~ithout great experimental effort to obtain fusion proteins according to the invention ~ith a small "ballast portion" and having particular desired properties.
Thus, if the desired protein is a eukaryotic protein the fusion proteins obtained according to the invention are composed exclusively or virtually exclusively of eukaryo-tic protein sequences. However, surprisingly, these fusion proteins are not recognized as foreign proteins by 133989~
-~ 4 - the prokaryotic host cells, and are not rapidly degraded by proteases intrinsic to the host. This degradation takes place particularly often in the case of proteins which are foreign to the host and coded for by cDNA sequences which are to be expressed in bacteria. It has now emerged that cDNA sequences can be expressed very effectively if they are "embedded" in the segments according to the invention.
It is possible to construct specific vectors for this pur-pose, which contain between the sequences according to the invention a polylinker sequence which has several cloning sites for the cDNA sequences. ~here the cDNA which has been cloned in contains no stop codon the polypeptide sequence coded for by the cDNA sequence is additionally protected by the polypeptide for which the C-terminal segment codes.
The fusion proteins can be cleaved chemically or enzymati-cally in a manner known per se. The choice of the suit-able method depends, in particular, on the amino acid sequence of the desired protein. If the latter contains, for example, no methionine it is possible for the connect-ing element to denote Met, in-which case chemical cleavage with cyanogen chloride or bromide is carried out. If there is a cysteine at the carboxyl terminal end of the connect-ing element, or if the connecting element represents Cys,then it is possible to carry out a cysteine-specific enzy-matic cleavage or chemical cleavage, for example after specific S-cyanylation. If there is a tryptophan at the carboxyl terminal end of the bridging element, or if the connecting element represents Trp, then chemical cleavage with N-bromosuccinimide can be carried out.
Desired proteins which do not contain Asp - Pro in their amino acid sequence and are sufficiently stable to acid can, as fusion proteins with this bridging element, be cleaved proteolytically in a manner known per se. This results in proteins which contain N-terminal proline or C-terminal aspartic acid. It is therefore also possible in this way to synthesize modified proteins.
1339~9~
The Asp-Pro bond can be made even more labile to acid if - this bridging element is (Asp)n-Pro or Glu-(Asp)n-Pro, n denoting 1-to 3.
Examples of enzymatic cleavages are likewise known, it also being possible to use modified enzymes of improved specificity (cf. C.S. Craik et al., Science 228 (1985) 291-297). If the desired eukaryotic peptide is proinsulin, then the chosen sequence is advantageously a peptide sequence in which an amino acid which can be split off with trypsin (Arg, Lys) is bonded to the N-terminal amino acid (Phe) of proinsulin, for example Ala-Ser-Met-Thr-Arg, since in this case the arginine-specific cleavage can be carried out with the protease trypsin.
If the desired protein does not contain the amino acid sequence Ile-Glu-Gly-Arg, then the fusion protein with the appropriate bridging element can be cleaved with factor Xa (EP-A 0,025,190 and 0,161,973).
The fusion protein is obtained by expression in a suit-able expression system in a manner known per se. All known host-vector systems are suitable for this purpose, that is to say, for example, mammalian cells and micro-organisms, for example yeasts and, preferably, bacteria, in particular E. coli.
The DNA sequence which codes for the desired protein is incorporated in a known manner into a vector ~hich ensures satisfactory expression in the selected expression system.
In bacterial hosts, it is advantageous to select the pro-moter and operator from the group comprising lac, tac, trp, PL or PR of phage ~, hsp, omp or a synthetic pro-moter as proposed in, for example, German Offenlegungs-schrift 3,430,683 (EP-A 0,173,149). The tac promoter-operator-sequence is advantageous and is now commercially - avai.able (for example expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equip-ment for Molecular Biology", 1984, page 63).
S In the expression of the fusion protein according to the invention it may prove advantageous to modify individual tripLets for the first few amino acids downstream of the ATG start codon in order to prevent any base-pairing at the mRNA level. Modifications of this type, as well as modifications, deletions or additions of individual amino acids in the IL-2 protein portion, are familiar to those skilled in the art, and the invention likewise relates to them. Elimination of cysteine or replaçement of cysteine by other amino acids, in order to prevent formation of undesired disulfide bridges, as is disclosed in, for ex-ample, EP-A 109,748, may be mentioned by way of example.
Figures 1 to 13 illustrate in the manner of a flow diagram the processes of the syntheses described in the examples having the same numbers. To facilitate comprehension, the preparation of the starting materials and intermediates has been depicted in Figures A to C. For the sake of clarity the reference numbers in Figures 1 to 13 each start a new decade, thus (11) in Figure 1. Reference numbers of starting materials to which the present appli-cation does not relate end with zero, thus, for example, (20) in Figure 2. The figures are not drawn to scale, in particular the scale is expanded appropriately in the region of the polylinker sequences. IL-2 sequences are defined by thick lines, and structural genes for desired proteins are emphasized in other ways.
Figure A gives an overview of the segments A to F accord-ing to the invention and of the combination of segments A and B. The starting material is the plasmid p159/6, whose preparation is described in detail in EP-A 0,163,249 and which is defined by Figure 5 in this publication.
Figure 3 shows the expression plasmid pE~1000, whose - 7 _ 13398~4 preparation is described in German Patent Application P 3,541,856.7 and is shown in Figure 1 therein. This plasmid is opened in the polylinker sequence by appropri-ate double digestion, this resulting in the linearized S plasmids (Ex1) to (Ex4).
Figure C shows the preparation of the pUC12 derivative p~226 and of the expression plasmid p~Z26-1, both of which contain segments A and F separated by a polylinker se-quence.
Figure 1 shows the preparation of the pUC12 derivativepKH40 and of the expression plasmid pK40, which code for fusion proteins in which the protein sequence correspond-ing to segment A, that is to say the first 22 amino acidsof IL-2, is followed by the bridging element Thr-Arg, with subsequently the amino acid sequence of proinsulin.
Figure 2 shows the construction of the plasmid pSL11 and of the expression plasmid pSL12, which code for polypep-tides in which the segment A is followed by a bridging element corresponding to polylinker sequences t2) and (20a), with subsequently the amino acid sequence of proinsulin.
Figure 3 shows the construction of the expression plasmid pK50 which codes for a polypeptide in which segments A
and B, that is to say the first 38 amino acids of IL-Z, are dlrectly followed by the amino acid sequence of pro-insul ln.
30Figure 4 shows the construction of the expression plasmid pKS1 which codes for a polypeptide in which segments A
and B are followed by a bridging element corresponding to sequences (42) and (41), to which is connected the amino acid sequence of proinsulin.
Figure 5 shows the construction of the expression plasmid pK52 which differs from pKS1 by the inserted MluI linker (51) which codes for the amino acid sequence which permits - - 8 - 1~3~9~
cleavage with factor Xa. pKS2 can also be obtained from pKS0 (Figure 3) by cleavage with MluI and introduction of the said MluI linker.
S Figure 6 shows the construction of the expression plasmid pK53 from pKS1 (Figure 4), likewise by introduction of the MluI linker.
Figure 7 shows the construction of the expression plasmid pSL14 from pSL12 (Figure 2) by introduction of the frag-ment C into the polylinker. This results in direct attach-ment of the segment C to the segment A. In the following polylinker the first two amino acids (each Glu) corre-spond to amino acids 60 and 61 of IL-2. Thus the IL-2 portion is composed of amino acids 1 to 22 and 37 to 61.
The subsequent amino acid sequence corresponds to that which is coded for by the plasmid pSL12 (Figure 2).
Figure 8 shows the construction of the expression plasmid pPH31 which codes for a fusion protein in which segments A to C are followed by a bridging element which is re-presented by sequence (81), with subsequently the amino acid sequence of proinsulin.
Figure 9 shows the construction of the plasmid pK192 which codes for a fusion protein in which segements A and B are followed by methionine and, thereafter, the amino acid sequence of hirudin.
Figure 10 shows the construction of the plasmid pW214 which codes for a fusion protein in which segments A and B are followed by the amino acid sequence which permits cleavage with factor Xa, with subsequently the amino acid sequence of granulocyte/macrophage colony stimulating factor (CSF).
Figure 11 shows the construction of the expression plasmid p~233 which codes for a fusion protein in which segments A and C (corresponding to amino acids 1 to 22 and 37 to 61 of IL-2) are followed by the bridging element Leu-Thr-Ile-Asp-Asp-Pro, with subsequently the amino acid sequence of CSF.
Figure 12 shows the construction of the expression plasmid p~234 which codes for a fusion protein having the follow-ing amino acid sequence: Segment A (amino acids 1 to 22) is followed by a bridging element Thr-Arg, then by segment D (amino acids 59 to 96 of IL-2), by Thr-Asp-Asp-Pro as a further connecting element, and finally by CSF.
Figure 13 shows the construction of the plasmids pH200 and pH201 and of the expression plasmid pH202. These plasmids have a polylinker located between segments A and F or A, P and F, into whose numerous cleavage sites foreign DNA can be cloned. These plasmids are particularly suitable for cloning cDNA sequences.
The invention is explained in detail in the examples which follow, in which the numbering coincides with that in the figures. Unless otherwise stated, percentage data relate to weight.
Example A
The starting plasmid p159/6 is described in EP-A 0,163,249 (Figure S). The sequence defined there as "IL-2" or in the text as "DNA sequence I" is in Figure A divided into segments A to F which are bounded by cleavage sites for the enzymes EcoRI, PstI, MluI, XbaI, SacI, PvuI and SalI.
Double digestion with the appropriate enzymes results in the segments (A) to (F) or adjoined segments, for example the segment (A,B) with EcoRI and MluI.
Example E
The preparation of the expression plasmid pEW1000 has been proposed in the (not prior-published) German Patent Appli-cation P3,541,856.7 (Figure 1). This plasmid is a derivative of the plasmid ptac11 (Amann et al., Gene 25 (1983) 167 - 178) into whose recognition site for EcoRI
has been incorporated a synthetic sequence which contains a SalI cleavage site. In this way the expression plasmid S pKK177.3 is obtained. Insertion of the lac repressor (Farabaugh, Nature 274 (1978) 765 - 769) results in the plasmid pJF118. This is opened at the unique restriction cleavage site for AvaI, and is, in a known manner, short-ened by about 1000 bp by exonuclease treatment and is ligated. This results in the plasmid pEW1000. Opening this plasmid in the polylinker using the enzymes EcoRI
and HindIII, SalI, PstI or SmaI results in the linearized expression plasmids (Ex1), (Ex2), (Ex3) and (Ex4).
Example C
The commercially available plasmid pUC12 is opened with EcoRI and SalI, and the linearized plasmid (1) is iso-lated. Ligation of (1) with the segment (A), the synthe-tic linker sequence (2) and the segment (F) results inthe plasmid p~226 (3).
The strain E. coli 79/02 is transformed in a known manner with the plasmid DNA from the ligation mixture. The cells are plated out on agar plates which contain isopropyl-B-D-thiogalactopyranoside (IPTG), S-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal) and 20 ~g/ml ampicillin (Ap).
The plasmid DNA is obtained from white clones, and the formation of the plasmid (3) is confirmed by restriction analysis and DNA sequence analysis.
The small EcoRI-HindIII fragment (4) is cut out of the plasmid (3) and is isolated. This fragment is ligated with the linearized expression plasmid (Ex1) in a T4 DNA
ligase reaction. The resulting plasmid pW226-1 (S) is characterized by restriction analysis.
Competent cells of the strain E. coli Mc 1061 are trans-formed with DNA from the plasmid pW 226-1. Clones which 133989~
are resistant to ampicillin are isolated on Ap-containing agar plates. The plasmid DNA is reisolated from Mc 1061 cells and then characterized anew by restriction analysis.
Competent cells of the E. coli strain W 3110 are now transformed with plasmid DNA isolated from E. coli Mc 1061 cells. E. coli ~ 3110 cells are always used for expression hereinafter. All the expression experiments in the stated examples are carried out in accordance with the following conditions.
An overnight culture of E. coli cells which contain the plasmid (5) is diluted in the ratio of about 1:100 with LB medium (J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, 1972) which contains 50 ~g/ml ampicillin, and growth is followed by measurement of the OD. ~hen the OD is 0.5 the culture is adjusted to 1 mM in IPTG and, after 150 to 180 minutes, the bacteria are spun down. The bacteria are boiled in a buffer mix-ture (7M urea, 0.1% SDS, 0.1 M sodium phosphate, pH 7.0) for 5 minutes, and samples are applied to a SDS gel electrophoresis plate. After electrophoresis, bacteria which contain the plasmid (5) produce a protein band which corresponds to the size of the expected protein (6 kD).
The stated induction conditions apply to shake cultures;
for larger fermentations appropriate modifications of the OD values and, where appropriate, slight variations in the IPTG concentrations are advantageous.
The resulting protein shows no biological activity in a cell proliferation test with an IL-2-dependent cell line (CTLL 2).
Example 1 The plasmid (3) is opened with MluI and SalI, and the two resulting fragments are separated by gel electrophoresis.
The larger fragment (11) is isolated.
- 12 - 1 ~ 3 9 ~ 9 4 The synthetic oligonucleotide (12) is ligated with the blunt-ended DNA (13) coding for proinsulin (Wetekam et al., ~ene 19 (1982) 179 - 183), this resulting in DNA sequence (14). The latter is cut with MluI and SalI, this result-ing in DNA sequence (15). The latter is now ligated witht~7e fragment (11), this resulting in formation of the pLasmid pKH40 (16). The latter is characterized by re-striction analysis.
The plasmid (16) is digested with EcoRI and HindIII, and the small fragment (17) is isolated by gel electrophoresis.
Ligation with the linearized expression plasmid (Ex1) re-sults in the expression plasmid pK40 (18). Expression as indicated in Example C results in a protein which, after cell disruption, is found in the soluble fraction of cellular protein. The Western blot technique is used to demonstrate that the proinsulin sequence is intact.
Example 2 The starting material is the plasmid pPH30 which is de-picted in the (not prior-published) German Patent Appli-cation P 3,541,856.7, in Figure 3c. Within the meaning of the present invention, in Figure 2 the IL-2 part-sequence is shown as "A-E" (20). The end of this se-quence and the bridging element up to the proinsulin se-quence is shown as (20a) in Figure 2.
The plasmid (20) is digested with PvuI and HindIII, and the smalL fragment (22) is isolated. In addition, the plasmid (3) is opened with EcoRI and PvuI, and the small fragment (23) is isolated. Moreover, the vector pUC12 is digested with EcoRI and HindIII, and the large fragment (21) is isolated. Ligation of the fragments (21), (23) and (22) results in the plasmid pSL11 (24).
The plasmid (24) is digested with HindIII and partially with EcoRI, and the fragment (25) which contains the segment A and the proinsulin gene is isolated. Ligation of (25) into the linearized expression plasmid (Ex1) re-sults in the expression plasmid pSL12 (26).
Expression as indicated in Example C and subsequent work-ing up results in a soluble fusion protein. ~estern blot analysis with insulin antibodies confirms that this pro-tein contains the intact insulin sequence.
Example 3 The plasmid ptrpED5-1 (30) (Hallewell et al., Gene 9 (1980) 27-47) is used for amplification of the proinsulin gene.
The plasmid is opened with HindIII and SalI, and the large fragment (31) is isolated. The fragment (31) is ligated with DNA sequence (14), this resulting in the plasmid pH106/4 (32).
The plasmid (32) is digested with SalI and MluI, and the small fragment (15) is isolated. The linearized expres-sion plasmid (Ex2), the segment (A,B) and the fragment(15) are now ligated, this resulting in the expression plasmid pK50 (33).
Expression of the coded fusion protein is carried out as indicated in Example C. The cells are then spun down from the culture broth and ruptured in a French press.
The protein suspension is now centrifuged to separate it into its soluble and insoluble protein constituents. The two fractions are analyzed by gel electrophoresis in a known manner on 17.5% SDS polyacrylamide gels and subse-quently by staining the proteins with the dyestuff Coo-massie blue. It is found, surprisingly, that the fusion protein is located in the insoluble sediment. Western blot analysis with insulin antibodies confirms that in-tact proinsulin is present in the fusion protein.
The sediment from the French press disruption can now im-mediately be used further for isolation of proinsulin.
- - 14 - I 3 3 9 8 9 ~
Example 4 The starting material is the plasmid pPH20 (40) which is depicted in German Patent Application P 3,541,856.7, in S Figure 3c. Cutting this plasmid with EcoRI, filling in the protruding ends and cutting with HindIII results in the fragment (41), from which the DNA sequence of the part of (40) which is of interest here can be seen.
Ligation of the linearized expression plasmid (Ex4) with the segment (A,B) the synthetic oligonucleotide (42) and the fragment (41) results in the plasmid pK51 (43).
Example 5 Ligation of the linearized expression plasmid (Ex2) with the segment (A,B), the synthetic oligonucleotide (51) and the DNA sequence (15) results in the plasmid pK52 (52).
The correct orientation of the oligonucleotide (51) is established by sequence analysis. The plasmid codes for a fusion protein which contains the amino acid sequence which corresponds to the oligonucleotide (51) and thus can be cleaved by activated Factor Xa.
The plasmid (52) can also be obtained in the following manner:
Partial cutting of the plasmid (33) with MluI and ligation of the resulting opened plasmid (53) with the DNA sequence (51) likewise results in the plasmid pK52.
Example 6 Partial cutting of the plasmid (43) with MluI and ligation of the resulting linearized plasmid (61) with the synthe-tic DNA sequence (51) results in the plasmid pK53 (62).The latter likewise codes for a fusion protein which can be cleaved with activated factor Xa. The correct orient-ation of the sequence (51) is established, as in Example 5, by DNA sequence analysis.
Example 7 The plasmid (26) is cleaved with XbaI and partially with MluI, and the large fragment (71) is isolated. Ligation with the segment (C) results in the plasmid pSL14 (72).
After expression and cell disruption, the fusion protein is found in the soluble fraction of cellular protein.
Example 8 The plasmid (20) is cleaved with XbaI and partially with EcoRI, and the protruding ends are fiLled in, this result-ing in DNA sequence (81). Ligation under blunt end con-ditions results in the plasmid pPH31 (82). The fusion protein is found in the insoluble fraction of cellular protein.
Example 9 The starting material used is the plasmid (90) which is described in EP-A 0,171,024 (Figure 3). This plasmid is - reacted with SalI and then with AccI, and the small frag-ment (91) is isolated. The latter is ligated with the synthetic oligonucleotide (92), this resulting in DNA
sequence (93). The latter is cut with MluI, this result-ing in DNA fragment (94).
The plasmid (33) is digested with MluI, partially, and ~ith SalI, and the large fragment (95) is isolated. The latter is ligated with the DNA sequence (94), this result-ing in the expression plasmid pK192 (96). The latter codes for a fusion protein in which the first 38 amino acids of IL-2 are followed by methionine and then by the amino acid sequence of hirudin. The fusion protein is found in the soluble fraction of cellular protein.
Example 10 The starting material used is the plasmid pHG23 (100) - 16 - I~ 3 9 8 9 ~
which is described in EP-A 0,183,350 and which is gener-ally accessible from the American Type Culture Collection under No. ATCC 39000. This plasmid is cut with SfaNI, the protruding ends are filled in, then reaction with PstI is carried out, and the small fragment (101) is iso-lated. Ligation of the linearized expression plasmid (Ex3) with the segment (A,~), the synthetic oligonucleo-tide (102) and the fragment (101) results in the express-ion plasmid p~214 (103). This plasmid codes for a fusion protein in which the first 38 amino acids of IL-2 are followed by the sequence which is derived from the oligo-nucleotide (102) and which allows the molecule to be cleaved with factor Xa, with subsequently the am;no acid sequence of CSF. After cell disruption, the fusion pro-tein is found in the insoluble fraction of cellular pro-tein.
Example 11 The starting plasmid p~216 (110) is proposed in German Patent Application P 3,545,568.3 (Figure 2b). In this plasmid, the IL-2 sequence corresponding to segments A
to E (PvuI cleavage site) is followed by a linker which codes for the amino acids Asp-Asp-Pro, immediately followed by the amino acid sequence for CSF. The connecting se-quence between IL-2 and CSF allows the fusion protein to be cleaved proteolytically.
The sequence (111) is isolated from the plasmid (110) by cutting with PvuI and HindIII.
The plasmid t3) is cut with MluI and XbaI, and the large fragment (112) is isolated. The latter is ligated with the segment (C), this resulting in the plasmid pW227 (113).
This plasmid is reacted with EcoRI and HindIII, and the short fragment (114) is isolated. If this fragment is ligated with the linearized expression plasmid (Ex1) the result is the plasmid p~227-1 (115). The plasmid codes for a protein which is derived from IL-2 but which has no 133989~
IL-2 activity.
The plasmid (113) is additionally cut ~ith EcoRI and Pvu~, and the short fragment (116) is isolated. Ligation of the linearized expression plasmid (Ex1) with the fragments (116) and (111) results in the expression plasmid pW233 (117). The latter codes for an insoluble fusion protein ~hich, by reason of the abovementioned linker, can be cleaved proteolytically.
Example 12 The plasmid (3) is cut with XbaI and SacI, and the large fragment (121) is isolated. Ligation with the segment (D) results in the plasmid pW228 (122). The latter is cut ~ith EcoRI and HindIII, and the small fragment (123) is isolated. Ligation of the linearized expression plas-mid (Ex1) ~ith the fragment (123) results in the express-ion plasmid pW228-1 (124). This plasmid codes for a bio-logically inactive IL-2 derivative. The plasmid is diges-ted ~ith EcoRI and PvuI, and the short fragment (125) is isolated. Ligation of the linearized expression plasmid (Ex1) with the fragments (125) and (111) results in the expression plasmid p~234 (126). The latter codes for a sparingly soluble fusion protein which can likewise be cleaved proteolytically.
Example 13 For the construction of plasmids which are suitable, in particular, for the expression of cDNA sequences, initially the polylinker sequence (131) is synthesized.
Ligation of the linearized plasmid (1) ~ith the segment (A), the polylinker sequence (131) and segment (F) results in the plasmid pH200 (132).
The plasmid (132) is reacted ~ith EcoRI and MluI, and the large fragment (133) is isolated. Ligation of the latter with the segment (A,B) results in the plasmid pH201 (134).
The plasmid (134) is reacted with EcoRI and HindIII, a~nd tne short fragment (135) is isolated. Ligation of this fragment with the linearized expression plasmid (Ex1) re-sults in the expression plasmid pH 202 (136).
The plasmid (136) is opened with BamHI, and the cDNA which is to be expressed is introduced into the linearized plas-mid via a commercially available BamHI adaptor. Depend-ing on the orientation of the cDNA, every third sequence is attached to (A,B) in the reading frame. If the cDNA
sequence contains no stop codon the polypeptide sequence for which it codes is additionally protected by the amino acid sequence corresponding to the segment (F).
If the cDNA is not connected in the correct reading frame, a shift of the reading frame is brought about by, for example, cleaving the cDNA-containing (original or multiplied) plasmids with MluI or XbaI (as long as the cDNA does not contain cleavage sites for these enzymes) and filling in the protruding ends by a Klenow polymerase reaction.
Addendum Triplet No. 0 1 ~ 2 Amino acid Met Ala Pro Nucleotide No. 1 10 Cod. strand 5 ' AA TTC ATG GCG CCG
Non-cod. strand 3' G TAC CGC GGC
(EcoRI) Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG
TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC
Gln Leu Glu His Leu Leu Leu Asp Leu Gln CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG
GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC
PstI
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys ATG ATC CTG AAC GGT ATC AAC AAC TAC AAA
TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC
TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG
MluI
Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA
TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT
Leu Lys His Leu Gln Cys Leu Glu Glu Glu CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG
GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC
XbaI
Leu Lys Pro Leu Glu Glu Val Leu Asn Leu CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG
GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC
Ala Gln Ser Lys Asn Phe His Leu Arg Pro GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG
CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC
GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG
Val Leu Glu Leu Lys Gly Ser Glu Thr Thr GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG
CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC
SacI
.
133989~
Phe Met Cys Glu Tyr Ala Asp Glu ~ Thr Ala TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG
AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC
TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG
PvuI
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu ACC TTC TGC CAG TCG ATC ATC TCT ACC CTG
TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC
Thr ACC TGA TAG 3 ' TG6 ACT ATC AGC T S' ( S a l I )
Claims (22)
1. A fusion protein which contains a C- or N-terminal portion which relates to segments corresponding to amino acids 1-22, 1-38, 37-61, 59-96 of Interleukin-2 (IL-2) or combinations thereof, and another portion selected from the group consisting of hirudin and proinsulin.
2. A fusion protein as claimed in claim 1, wherein the said amino acid sequence corresponds to that of human IL-2.
3. A fusion protein claimed in claim 1, wherein the gene coding for IL-2 contains the parts of DNA sequence I
Triplet No. 0 1 2 Amino acid Met Ala Pro Nucleotide No. 1 10 Cod. strand 5' AA TTC ATG GCG CCG
Non-cod. strand 3' G TAC CGC GGC
(EcoRI) Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG
TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC
Gln Leu Glu His Leu Leu Leu Asp Leu Gln CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG
GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC
PstI
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys ATG ATC CTG AAC GGT ATC AAC AAC TAC AAA
TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC
TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG
MluI
Lys Phe Tyr Met Pro Lys Lys Ala Ihr Glu AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA
TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT
Leu Lys His Leu Gln Cys Leu Glu Glu Glu CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG
GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC
XbaI
Leu Lys Pro Leu Glu Glu Yal Leu Asn Leu CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG
GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC
Ala Gln Ser Lys Asn Phe His Leu Arg Pro GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG
CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC
GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG
Val Leu Glu Leu Lys Gly Ser Glu Thr Thr GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG
CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC
SacI
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG
AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC
TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG
PvuI
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu ACC TTC TGC CAG TCG ATC ATC TCT ACC CTG
TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC
Thr ACC TGA TAG 3' TGG ACT ATC AGC T 5' (SalI) as described in claim 1.
Triplet No. 0 1 2 Amino acid Met Ala Pro Nucleotide No. 1 10 Cod. strand 5' AA TTC ATG GCG CCG
Non-cod. strand 3' G TAC CGC GGC
(EcoRI) Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu ACC TCT TCT TCT ACC AAA AAG ACT CAA CTG
TGG AGA AGA AGA TGG TTT TTC TGA GTT GAC
Gln Leu Glu His Leu Leu Leu Asp Leu Gln CAA CTG GAA CAC CTG CTG CTG GAC CTG CAG
GTT GAC CTT GTG GAC GAC GAC CTG GAC GTC
PstI
Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys ATG ATC CTG AAC GGT ATC AAC AAC TAC AAA
TAC TAG GAC TTG CCA TAG TTG TTG ATG TTT
Asn Pro Lys Leu Thr Arg Met Leu Thr Phe AAC CCG AAA CTG ACG CGT ATG CTG ACC TTC
TTG GGC TTT GAC TGC GCA TAC GAC TGG AAG
MluI
Lys Phe Tyr Met Pro Lys Lys Ala Ihr Glu AAA TTC TAC ATG CCG AAA AAA GCT ACC GAA
TTT AAG ATG TAC GGC TTT TTT CGA TGG CTT
Leu Lys His Leu Gln Cys Leu Glu Glu Glu CTG AAA CAC CTC CAG TGT CTA GAA GAA GAG
GAC TTT GTG GAG GTC ACA GAT CTT CTT CTC
XbaI
Leu Lys Pro Leu Glu Glu Yal Leu Asn Leu CTG AAA CCG CTG GAG GAA GTT CTG AAC CTG
GAC TTT GGC GAC CTC CTT CAA GAC TTG GAC
Ala Gln Ser Lys Asn Phe His Leu Arg Pro GCT CAG TCT AAA AAT TTC CAC CTG CGT CCG
CGA GTC AGA TTT TTA AAG GTG GAC GCA GGC
Arg Asp Leu Ile Ser Asn Ile Asn Val Ile CGT GAC CTG ATC TCT AAC ATC AAC GTT ATC
GCA CTG GAC TAG AGA TTG TAG TTG CAA TAG
Val Leu Glu Leu Lys Gly Ser Glu Thr Thr GTT CTG GAG CTC AAA GGT TCT GAA ACC ACG
CAA GAC CTC GAG TTT CCA AGA CTT TGG TGC
SacI
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala TTC ATG TGC GAA TAC GCG GAC GAA ACT GCG
AAG TAC ACG CTT ATG CGC CTG CTT TGA CGC
Thr Ile Val Glu Phe Leu Asn Arg Trp Ile ACG ATC GTT GAA TTT CTG AAC CGT TGG ATC
TGC TAG CAA CTT AAA GAC TTG GCA ACC TAG
PvuI
Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu ACC TTC TGC CAG TCG ATC ATC TCT ACC CTG
TGG AAG ACG GTC AGC TAG TAG AGA TGG GAC
Thr ACC TGA TAG 3' TGG ACT ATC AGC T 5' (SalI) as described in claim 1.
4. A fusion protein as claimed in claim 3, wherein the gene coding for the IL-2 portion is essentially composed of 1, 2 or 3 of the segments A to F of the IL-2 gene (EcoRI)-A-PstI-B-MluI-C-XbaI-D-SacI-E-PvuI-F-(SalI) in arbitrary sequence.
5. The fusion protein as claimed in claim 4 wherein the segments are linked via adaptor or linker sequences.
6. The fusion protein as claimed in claim 4, wherein segments A to F are as shown in Figure A.
7. A fusion protein as claimed in claim 1, wherein between the IL-2 sequence and the amino acid sequence of the desired protein, is located an amino acid or amino acid sequence selected from Met, Cys, Trp, Lys, Arg, Asp-Pro, and Ile-Glu-Gly-Arg, which allows the desired protein to be cleaved off chemically or enzymatically, from the IL-2 position.
8. A fusion protein as claimed in claim 7, wherein the amino acid is Met, Cys, Trp, Lys or Arg, or the amino acid sequence contains these amino acids at the C-terminal end.
9. A fusion protein as claimed in claim 8, wherein the amino acid sequence is Asp-Pro or contains this amino acid sequence at the C-terminal end.
10. A fusion protein as claimed in claim 7, wherein the amino acid sequence is Ile-Glu-Gly-Arg or contains this amino acid sequence at the C-terminal end.
11. A process for the preparation of a fusion protein as claimed in claim 1, which comprises causing the expression of a gene coding for the fusion protein in a host cell.
12. A process for the preparation of a fusion protein as claimed in claim 2, which comprises causing the expression of a gene coding for the fusion protein in a host cell.
13. The process as claimed in claim 11, wherein the gene is incorporated in an expression vector and is expressed in a bacterial cell.
14. The process as claimed in claim 12, wherein the gene is incorporated in an expression vector and is expressed in a bacterial cell.
15. The process as claimed in claim 13 or 14, wherein the bacterial cell used is E. coli.
16. A gene structure coding for a fusion protein as claimed in claim 1.
17. A gene structure coding for a fusion protein as claimed in claim 2.
18. A plasmid containing a gene structure as claimed in claim 16.
19. A plasmid containing a gene structure as claimed in claim 17.
20. Plasmids pW226, pW226-1, pK40, pSC12, pK50, pK51, pK52, pK53, pS214, pPH31, pK192, pW214, pW227, pW227-1, pW233, pW228, pW228-1, pW234, pH200, pH201, pH202.
21. A host cell containing a vector as claimed in claim 18, 19 or 20.
22. The use of the fusion protein as claimed in any one of claims 1 to 10 for the preparation of the desired protein.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3545565 | 1985-12-21 | ||
| DEP3545565.9 | 1985-12-21 | ||
| DEP3636903.9 | 1986-10-30 | ||
| DE19863636903 DE3636903A1 (en) | 1985-12-21 | 1986-10-30 | FUSION PROTEINS WITH EUKARYOTIC BALLASTES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1339894C true CA1339894C (en) | 1998-06-02 |
Family
ID=25839208
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000525858A Expired - Lifetime CA1339894C (en) | 1985-12-21 | 1986-12-19 | Fusion proteins with a eukaryotic ballast portion |
Country Status (16)
| Country | Link |
|---|---|
| EP (1) | EP0229998B1 (en) |
| JP (2) | JP2553058B2 (en) |
| KR (1) | KR950000301B1 (en) |
| AT (1) | ATE78296T1 (en) |
| AU (1) | AU599943B2 (en) |
| CA (1) | CA1339894C (en) |
| DE (1) | DE3636903A1 (en) |
| DK (1) | DK168823B1 (en) |
| ES (1) | ES2033678T3 (en) |
| FI (1) | FI93734C (en) |
| GR (1) | GR3005985T3 (en) |
| HU (1) | HU209747B (en) |
| IE (1) | IE58935B1 (en) |
| IL (1) | IL81018A (en) |
| NO (1) | NO175003C (en) |
| PT (1) | PT83973B (en) |
Families Citing this family (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3545568A1 (en) * | 1985-12-21 | 1987-07-16 | Hoechst Ag | GM-CSF-PROTEIN, ITS DERIVATIVES, PRODUCTION OF SUCH PROTEINS AND THEIR USE |
| DE3844211A1 (en) * | 1988-12-29 | 1990-07-05 | Hoechst Ag | NEW INSULINE DERIVATIVES, THE PROCESS FOR THEIR PRODUCTION, THEIR USE AND A PHARMACEUTICAL PREPARATION CONTAINING THEM |
| FR2643646B1 (en) * | 1989-02-27 | 1993-09-17 | Pasteur Institut | EXPRESSION OF NUCLEOTIDES SEQUENCES ENCODING FOR GAS VESICLES |
| CU22222A1 (en) * | 1989-08-03 | 1995-01-31 | Cigb | PROCEDURE FOR THE EXPRESSION OF HETEROLOGICAL PROTEINS PRODUCED IN A FUSION FORM IN ESCHERICHIA COLI, ITS USE, EXPRESSION VECTORS AND RECOMBINANT STRAINS |
| DE3942580A1 (en) * | 1989-12-22 | 1991-06-27 | Basf Ag | METHOD FOR PRODUCING HIRUDINE |
| DE4105480A1 (en) * | 1991-02-21 | 1992-08-27 | Boehringer Mannheim Gmbh | IMPROVED ACTIVATION OF RECOMBINANT PROTEINS |
| DK0821006T3 (en) | 1996-07-26 | 2004-08-16 | Aventis Pharma Gmbh | Insulin derivatives with increased zinc binding |
| DE19825447A1 (en) | 1998-06-06 | 1999-12-09 | Hoechst Marion Roussel De Gmbh | New insulin analogues with increased zinc formation |
| DE102006031955A1 (en) | 2006-07-11 | 2008-01-17 | Sanofi-Aventis Deutschland Gmbh | Process for the preparation of dibasic B chain end insulin analogs |
| DE102006031962A1 (en) | 2006-07-11 | 2008-01-17 | Sanofi-Aventis Deutschland Gmbh | Amidated insulin glargine |
| KR101820024B1 (en) | 2008-10-17 | 2018-01-18 | 사노피-아벤티스 도이칠란트 게엠베하 | Combination of an insulin and a GLP-1 agonist |
| HUE037735T2 (en) | 2009-11-13 | 2018-09-28 | Sanofi Aventis Deutschland | PHARMACEUTICAL COMPOSITION COMPRISING desPro36Exendin-4(1-39)-Lys6-NH2 AND METHIONINE |
| ES2534191T3 (en) | 2009-11-13 | 2015-04-20 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical composition comprising a GLP-1 agonist, an insulin and methionine |
| BR112013004756B1 (en) | 2010-08-30 | 2020-04-28 | Sanofi Aventis Deutschland | use of ave0010 for the manufacture of a medication for the treatment of type 2 diabetes mellitus |
| US9821032B2 (en) | 2011-05-13 | 2017-11-21 | Sanofi-Aventis Deutschland Gmbh | Pharmaceutical combination for improving glycemic control as add-on therapy to basal insulin |
| RU2650616C2 (en) | 2011-08-29 | 2018-04-16 | Санофи-Авентис Дойчланд Гмбх | Pharmaceutical combination for use in glycemic control in patients with type 2 diabetes mellitus |
| TWI559929B (en) | 2011-09-01 | 2016-12-01 | Sanofi Aventis Deutschland | Pharmaceutical composition for use in the treatment of a neurodegenerative disease |
| JP6970615B2 (en) | 2014-12-12 | 2021-11-24 | サノフィ−アベンティス・ドイチュラント・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング | Insulin glargine / lixisenatide fixed ratio prescription |
| TWI748945B (en) | 2015-03-13 | 2021-12-11 | 德商賽諾菲阿凡提斯德意志有限公司 | Treatment type 2 diabetes mellitus patients |
| TW201705975A (en) | 2015-03-18 | 2017-02-16 | 賽諾菲阿凡提斯德意志有限公司 | Treatment of type 2 diabetes mellitus patients |
| KR20200080748A (en) | 2018-12-27 | 2020-07-07 | 주식회사 폴루스 | A Method for Purifying Proinsulin Using Anion Exchange Chromatography |
| KR20200080747A (en) | 2018-12-27 | 2020-07-07 | 주식회사 폴루스 | An Enzymatic Conversion Composition for Producing Insulin from Proinsulin and a Method for Producing Insulin from Proinsulin Using the Same |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK108685A (en) * | 1984-03-19 | 1985-09-20 | Fujisawa Pharmaceutical Co | GROWTH FACTOR I |
| EP0158198A1 (en) * | 1984-03-29 | 1985-10-16 | Takeda Chemical Industries, Ltd. | DNA and use thereof |
| GB8412517D0 (en) * | 1984-05-16 | 1984-06-20 | Nagai K | Recombinant fusion proteins |
-
1986
- 1986-10-30 DE DE19863636903 patent/DE3636903A1/en not_active Withdrawn
- 1986-12-16 ES ES198686117481T patent/ES2033678T3/en not_active Expired - Lifetime
- 1986-12-16 AT AT86117481T patent/ATE78296T1/en not_active IP Right Cessation
- 1986-12-16 EP EP86117481A patent/EP0229998B1/en not_active Expired - Lifetime
- 1986-12-18 FI FI865187A patent/FI93734C/en not_active IP Right Cessation
- 1986-12-18 IL IL81018A patent/IL81018A/en not_active IP Right Cessation
- 1986-12-19 HU HU865354A patent/HU209747B/en unknown
- 1986-12-19 PT PT83973A patent/PT83973B/en unknown
- 1986-12-19 NO NO865192A patent/NO175003C/en not_active IP Right Cessation
- 1986-12-19 CA CA000525858A patent/CA1339894C/en not_active Expired - Lifetime
- 1986-12-19 DK DK619186A patent/DK168823B1/en not_active IP Right Cessation
- 1986-12-19 IE IE333486A patent/IE58935B1/en not_active IP Right Cessation
- 1986-12-19 AU AU66760/86A patent/AU599943B2/en not_active Expired
- 1986-12-20 KR KR1019860011010A patent/KR950000301B1/en not_active IP Right Cessation
- 1986-12-22 JP JP61306185A patent/JP2553058B2/en not_active Expired - Lifetime
-
1992
- 1992-10-15 GR GR920402187T patent/GR3005985T3/el unknown
-
1995
- 1995-10-11 JP JP7263310A patent/JP2774260B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| HU209747B (en) | 1994-10-28 |
| FI865187A (en) | 1987-06-22 |
| EP0229998A2 (en) | 1987-07-29 |
| IE863334L (en) | 1987-06-21 |
| NO865192D0 (en) | 1986-12-19 |
| ES2033678T3 (en) | 1993-04-01 |
| EP0229998B1 (en) | 1992-07-15 |
| AU599943B2 (en) | 1990-08-02 |
| DK168823B1 (en) | 1994-06-20 |
| ATE78296T1 (en) | 1992-08-15 |
| JP2553058B2 (en) | 1996-11-13 |
| GR3005985T3 (en) | 1993-06-07 |
| IE58935B1 (en) | 1993-12-01 |
| PT83973B (en) | 1989-07-31 |
| DK619186D0 (en) | 1986-12-19 |
| DK619186A (en) | 1987-06-22 |
| JP2774260B2 (en) | 1998-07-09 |
| NO175003B (en) | 1994-05-09 |
| KR870006187A (en) | 1987-07-09 |
| NO175003C (en) | 1994-08-17 |
| FI865187A0 (en) | 1986-12-18 |
| IL81018A0 (en) | 1987-03-31 |
| JPH08187089A (en) | 1996-07-23 |
| AU6676086A (en) | 1987-06-25 |
| EP0229998A3 (en) | 1988-08-03 |
| NO865192L (en) | 1987-06-22 |
| FI93734C (en) | 1995-05-26 |
| HUT44614A (en) | 1988-03-28 |
| FI93734B (en) | 1995-02-15 |
| JPS62167799A (en) | 1987-07-24 |
| PT83973A (en) | 1987-01-01 |
| DE3636903A1 (en) | 1987-07-02 |
| IL81018A (en) | 1992-03-29 |
| KR950000301B1 (en) | 1995-01-13 |
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