| PATENT ASSIGNEE'S COUNTRY | USA |
| UPDATE | 05.00 |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 02.05.00 |
| PATENT TITLE |
Process for the production biologically active dimeric protein |
| PATENT ABSTRACT |
The present invention relates to a folding process for the preparation of biologically active, dimeric TGF-.beta. (Tranforming Growth Factor type .beta.)-like protein. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | 28.07.98 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED | This data is not available for free |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
I claim: 1. Process for the production of a dimeric, biologically active Transforming Growth Factor type .beta.3 (TGF-.beta.3) or a salt thereof, comprising treating the denatured monomeric form of said TGF-.beta.3 with a folding buffer comprising a mild detergent which permits folding of the monomeric TGF-.beta.3 into the spatial conformation which after dimerization is associated with the biological activity, while retaining said monomer in a soluble form, and an organic solvent selected from the group consisting of DMSO (Dimethylsufoxide), DMSO.sub.2 (Dimethylsulfone) and DMF (Dimethylformamide); and any mixture of two or three members of the group consisting of DMSO, DMSO.sub.2 and DMF. 2. The process according to claim 1, in which the buffer additionally contains a reducing substance. 3. The process according to claim 1, in which the mild detergent is selected from the group consisting of digitonin, 3-(3-chlolamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS), chlolamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO), and any mixture of the members of the group consisting of digitonin, CHAPS, and CHAPSO. 4. The process according to claim 3 in which the mild detergent is selected from the group consisting of CHAPS, CHAPSO and any mixture thereof. 5. The process according to claim 1 in which mild detergent is present in the folding buffer at a concentration of about 1 mM to 100 mM. 6. The process according to claim 1 in which mild detergent is present in the folding buffer at a concentration of 30 mM to 60 mM. 7. The process according to claim 1 in which mild detergent is present in the folding buffer at a concentration of 30 mM. 8. The process according to claim 1 in which the organic solvent is selected from the group consisting of DMSO, DMSO.sub.2, and DMF. 9. The process according to claim 1 in which organic solvent is used at a concentration from about 5% to about 40%, (vol/vol). 10. The process according to claim 9 in which organic solvent is used at a concentration from about 10% to about 30% (vol/vol). 11. The process according to claim 9 in which DMSO is used at a concentration of about 10% to about 30% (vol/vol). 12. The process according to claim 9 in which DMSO is used at a concentration of about 30% (vol/vol). 13. The process according to claim 9 in which DMF is used at a concentration of about 10% to about 30% (vol/vol). 14. The process according to claim 9 in which DMF is used at a concentration of about 10% (vol/vol). 15. The process according to claim 9 in which DMSO.sub.2 is used at a concentration of about 10% (vol/vol). 16. The process according to claim 9 wherein the organic solvent is a mixture of DMSO and DMF and the mixture is used in a concentration of 10 to 30% (vol/vol). 17. The process according to claim 1 in which the buffer has a pH of about 6 to about 10. 18. The process according to claim 1 in which the buffer has a temperature of about 0.degree. C. to about 40.degree. C. 19. The process according to claim 2 in which the reducing substance is a reduced sulfhydryl compound. 20. The process according to claim 19 in which the reduced sulfhydryl compound is selected from the group consisting of glutathione in its reduced form .beta.-mercaptoethanol in its reduced form, mercaptomethanol in its reduced form, cysteine, cysteamine, and dithiothreitol in its reduced form. 21. The process according to claim 20 in which the reduced sulfhydryl compound is used in a concentration of about 1 mM to 100 mM. 22. The process according to claim 20 in which the reduced sulfhydryl compound is used in a concentration of about 1 to 10 mM. 23. The process according to claim 20 in which the reduced sulfhydryl compound is used in a concentration of about 2.5 mM. |
| PATENT DESCRIPTION |
The present invention relates to a folding process for the preparation of biologically active, dimeric TGF-.beta. (Transforming Growth Factor type .beta.)-like protein. BACKGROUND OF THE INVENTION TGF-.beta.-like proteins, i.e. proteins of the TGF-.beta. superfamily, play a central role in many biological regulation pathways such as embryonal development or regeneration of tissue. They are very potent biological agents which can be used also therapeutically for a series of different purposes. The best known members of the TGF-.beta. superfamily are the TGF-.beta.s themselves. TGF-.beta. was originally purified to homogeneity from human platelets, human placenta and bovine kidney and identified as a homodimeric protein with a molecular mass of about 25.000 Da. First characterized by its ability to act synergistically with EGF or TGF-.alpha. to induce anchorage-independent growth of untransformed NRK cells, recently, TGF-.beta. has been shown to exhibit numerous regulatory effects on a wide variety of both normal and neoplastic cells indicating the importance of this protein as a multifunctional regulator of cellular activity. Depending upon the cell or tissue type, and the presence or absence of other growth factors, TGF-.beta. may either stimulate mitogenesis, cell proliferation and growth, or may effectively inhibit said processes, or may exhibit other actions like e.g. control of adipogenesis, myogenesis, chondrogenesis, osteogenesis and immune cell function, stimulation of chemotaxis, or induction or inhibition of differentiation. Many of the actions of TGF-.beta. are related to the response of cells or tissues to stress or injury, and to the repair of resultant damage. After inflammation, TGF-.beta. plays the major role in the formation of granulation tissue, increases the expression of genes associated with extracellular matrix formation such as fibronectin, collagen and several protease inhibitors and stimulates collagen-matrix contraction by fibroblasts, suggesting its possible role in connective tissue contraction. Until now, five distinct but functionally and structurally closely related TGF-.beta.s designated as TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4 and TGF-.beta.5 are described. All TGF-.beta.s are synthesized as 390 to 412 amino acid precursors that undergo proteolytic cleavage to produce the mature forms, which consist of the C-terminal 112 amino acids. In their mature, biologically active forms, TGF-.beta.1 to 5 are acid- and heat-stable disulfide-linked homodimers of two polypeptide chains of 112 amino acids each. The complete amino acid sequences of human (Derynck, R. et al. (1985) Nature 316, 701-705), murine (Derynck, R. et al. (1986) J. Biol. Chem. 261, 4377-4379) and simian TGF-.beta.1 (Sharples, K. et al. (1987) DNA 6, 239-244) show remarkable sequence conservation, differing only in a single amino acid residue. Comparison of the amino acid sequence of human TGF-.beta.1, human TGF-.beta.2 (deMartin, R. et al. (1987) EMBO J. 6, 3673-3677; Marquardt, H. et al. (1987) J. Biol. Chem. 262,12127-12131) and human TGF-.beta.3 (Ten Dijke, P. et al. (1988) PNAS 85, 4715-4719) has demonstrated that the three proteins exhibit in their mature forms about 70-80% sequence identity. A heterodimeric TGF-.beta.1.2 has been isolated from porcine platelets and consists of one subunit of TGF-.beta.1 disulfide-linked to one subunit of TGF-.beta.2 (Cheifetz, S. et al. (1987) Cell 48, 409-415). Recently, attempts have been undertaken aiming to produce TGF-.beta.s by means of recombinant techniques rather than isolating these factors from natural sources (e.g. platelets) in order to obtain sufficient amounts for testing in various therapeutic modalities. However, it has proven to be extremely difficult to obtain biologically active recombinant TGF-.beta.. As can be seen from the sequences depicted in the sequence listing under SEQ ID NOs.1 to 6, the 112 amino acids long mature forms of TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3 contain 9 cysteine residues. As has been shown for TGF-.beta.2 the 9 cysteine residues are forming 4 intrachain and 1 interchain disulfide bonds [Schlunegger, M. P. and Gruetter, M. G., Nature 358:430-434(1992)]. Heterologous expression of TGF-.beta. may lead to a product which, although having the correct primary structure, fails to fold properly to produce the correct, complicated secondary or tertiary structures and which, therefore, lacks the biological activity. Taking the complexity of the native TGF-.beta. molecules into account, it has generally been considered expedient to express the respective TGF-.beta. genes in cells derived from higher organisms. Although expression of recombinant TGF-.beta.s can be achieved in eukaryotic systems, the yields of biologically active, correctly folded material obtained are still far from being satisfactory. Therefore, attempts were made to produce biologically active TGF-.beta. in a microbial host. However, in e.g. bacteria the intracellular conditions are not conducive to correct folding, disulfide bond formation and disulfide-stabilized dimerization which is apparently essential for activity. Thus, only very little biologically active TGF-.beta. could be obtained after expression of the respective gene in E. coli under the control of the lambda promoter as described in European Patent Application EP-A-0 268 561. Another report describes the expression of a TGF-.beta. cDNA in E. coli under the control of the trp promoter yielding a radioactively labelled protein band with an apparent molecular weight of 13'000 Da in an autoradiogram of a SDS polyacrylamide gel, but no activity was measured (Urushizaki, Y. et al. (1987) Tumor Res. 22, 41-55). When recombinant proteins are produced at high levels in bacterial (such as E. coli) expression systems, they often appear in the form of highly insoluble intracellular precipitates referred to as inclusion bodies or refractile bodies which can be recognized as bright spots visible within the enclosure of the cells under a phase contrast microscope. These inclusion bodies, which can readily be separated from the soluble bacterial proteins, contain the recombinant protein in a mostly denatured and reduced form which does not exhibit the functional activity of its natural counterpart and which therefore is useless as a commercial product. It is therefore generally agreed, that the recombinant retractile protein has to be solubilized under conditions which are suitable in maintaining it in its denatured form and subsequently has to be folded in order to undergo the transition from the denatured unfolded form to the proper, functionally active three-dimensional structure, the conformation of which is stabilized by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions. In the case of cysteine containing proteins this process may also involve formation of disulfide bonds. When the formation of disulfide bonds is chemically promoted, the formation of incorrect intramolecular and, in the case of dimeric or multimeric proteins, intermolecular bridges should be prevented or at least minimized, since the formation of undesired, incorrectly folded isomers may yield nonhomogenous material, thus complicating the further purification of the protein having the desired structure, or may generate a protein with reduced activity. Folding of proteins usually is performed in a multistep process comprising the solubilization of the protein under strongly denaturing conditions, and then reducing the concentration of the chaotrop in order to allow the folding of the protein. However, such an approach failed in the folding of TGF-.beta.. However, in the European patent application EP-A-0 433 225 a successful process for the production of biologically active, dimeric TGF-.beta.-like protein is described, in which a mild detergent is used which allows the folding of the TGF-.beta. protein while the detergent remains present in the folding buffer. It is known from the prior art (Tam et al., J. Am. Chem. Soc. 113:6657-6662, 1991) that dimethyl sulfoxide (DMSO) can be used for promoting a selective and efficient formation of disulfide bonds in peptides. The method is selective, i.e. without side reactions, and a wide pH range can be applied. However, correct disulfide bridge formation was shown only for peptides up to about 30 amino acids. In another publication (Bentle et al., U.S. Pat. No. 4,731,440) dimethylsulfone or a mixture of dimethylsulfone and urea was used for solubilization of somatotropin from inclusion bodies. The solubilized protein then could be renatured by contacting the dimethylsulfone containing solution of the protein with a mild oxidizing agent. Surprisingly it was now found that a process in which a mild detergent is used for the folding of a TGF-.beta.-like protein can be improved if dimethyl sulfoxide (DMSO), dimethylsulfone (DMSO.sub.2) or dimethyl formamide (DMF) is added. OBJECT OF THE INVENTION It is the object of the present invention to provide an improved process for the production of biologically active, dimeric TGF-.beta.-like protein from its denatured or otherwise non-native form. This object is achieved by the unexpected finding that considerable amounts of the desired dimeric product can be obtained in an unexpected yield when the monomeric form of said protein is treated with a folding buffer which comprises (a) a mild detergent and (b) DMSO, DMF or DMSO.sub.2 or a mixture of two or three of the group consisting of DMSO, DMSO.sub.2 and DMF. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved process for the production of a dimeric, biologically active protein of a Transforming Growth Factor type .beta. (TGF-.beta.)-like protein, comprising treating a TGF-.beta.-like protein with a folding buffer comprising (a) a mild detergent which allows the folding of a protein of the TGF-.beta.-superfamily, and (b) an organic solvent selected from the group consisting of DMSO, DMF and DMSO.sub.2 and any mixture of two or three of the group consisting of DMSO, DMSO.sub.2 and DMF. The term "TGF-.beta.-like protein" in context with the present invention means a protein having in its monomeric form a sequence with at least 75% homology to at least one of the amino acid sequences of a monomer of the following members of the TGF-.beta. superfamily (which also fall within the term "TGF-.beta.-like protein"): TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3; a growth inhibitor isolated from conditioned medium of BSC-1 monkey kidney cells (i.e. polyergin; Holley, R. W. et al. (1980) PNAS 77, 5989-5992; Ristow, H. J. (1986) PNAS 83, 5531-5533); TGF-.beta.4 from chicken embryo chondrocytes (Jakowlew, S. B. et al. (1988) Molecular Endocrinology2, 1186-1195); TGF-.beta.5 from Xenopus-Laevis (Kondaiah, P. et al. (1990) J. Biol. Chem. 265, 1089-1093); TGF-.beta.-related inhibins and activins (gonadal proteins that regulate pituitary secretion of follicle stimulating hormone); Mullerian inhibiting substance (MIS, which inhibits the development of the Mullerian duct in mammalian male embryos); bone morphogenic proteins (BMP, a group of polypeptides involved in the induction of cartilage and bone formation; the members of this group known today are BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9); the transcript from the decapentaplegic gene complex of Drosophila (dpp, which acts to control morphogenesis in the fly embryo); Vg-1 (the product of the Xenopus transcript which is present in the vegetal pole of oocytes); and Vgr-1, a Vg-1 related mammalian gene (Mason, A. et al. (1986) Biochem. Biophys. Res. Commun. 135, 957-964; Cate, R. et al. (1986) Cell 45, 685-698; Wozney, J. M. et al. (1988) Science 242, 1528-1534; Padgett, R. et al. (1986) Nature 325, 81-84; Weeks, D. L. and Melton, D. A. (1987) Cell 51, 861-868; Lyons, K. et al. (1989) PNAS 86, 4554-4558). Also included within the meaning of "TGF-.beta.-like protein" are heterodimers containing subunits of different TGF-.beta. like proteins, or fragments or mutants of the above mentioned proteins which retain one or all of the biological activities of the parent molecule. In a preferred meaning the term "TGF-.beta.-like protein" in context with the present invention represents any protein of the TGF-.beta. superfamily. In a more preferred meaning it represents the following proteins of the TGF-.beta. superfamily: TGF-.beta.1, TGF-.beta.2 and TGF-.beta.3 of mammalian such as human or animal origin, e.g. simian, murine, porcine, equine or bovine, as well as heterodimeric TGF-.beta.s consisting of two different subunits of 112 amino acids each, and fragments and mutants of a TGF-.beta. including hybrid molecules in which parts of different TGF-.beta.s are exchanged; a growth inhibitor isolated from conditioned medium of BSC-1 monkey kidney cells (i.e. polyergin); TGF-.beta.4 from chicken embryo chondrocytes; TGF-.beta.5 from Xenopus-Laevis; TGF-.beta.-related inhibins and activins (gonadal proteins that regulate pituitary secretion of follicle stimulating hormone); Mullerian inhibiting substance (MIS, which inhibits the development of the Mullerian duct in mammalian mate embryos); bone morphogenic proteins (BMP, a group of polypeptides involved in the induction of cartilage and bone formation; the members of this group known today are BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8 and BMP-9); the transcript from the decapentaplegic gene complex of Drosophila (dpp, which acts to control morphogenesis in the fly embryo); Vg-1 (the product of the Xenopus transcript which is present in the vegetal pole of oocytes); and Vgr-1, a Vg-1 related mammalian gene. Also included within the meaning of "TGF-.beta.-like protein" are heterodimers containing subunits of different TGF-.beta. like proteins, or fragments and mutants of the above mentioned proteins which retain one or all of the biological activities of the parent molecule. Even more preferred TGF-.beta.-like proteins are selected from the group consisting of TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, heterodimeric TGF-.beta.s, fragments and mutants of a TGF-.beta. including hybrid molecules in which parts of different TGF-.beta.s are exchanged, BMPs, inhibins and activins. Even more preferred are those selected from the group consisting of BMP-2, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, and heterodimers and fragments and mutants thereof including hybrid molecules in which parts of the different TGF-.beta.s are exchanged, preferably TGF-.beta.1-3, TGF-.beta.2-3 or TGF-.beta.3-2 defined hereinafter or TGF-.beta.1-2 consisting, in N- to C-terminal order, of the N-terminal 44 amino acids of human TGF-.beta.1 and of the C-terminal 68 amino acids of TGF-.beta.2. Even more preferred TGF-.beta.-like proteins are those selected from the group consisting of the proteins having the amino acid sequences depicted in the sequence listing under SEQID No.1,3, 5, 7, 9, 11 or 13. The most preferred TGF-.beta.-like protein is TGF-.beta.3. The term BMP-2 includes also variants of BMP-2 which have the same biological activity. Such variants are e.g. those prepared in the Examples, i.e. the BMP-2 of SEQ ID NO.13 with an additional N-terminal methionine and the BMP-2 of SEQ ID NO.13 lacking the N-terminal amino acid Gln. The biological activity of TGF-.beta. for the purpose herein is defined as either the cell migration promoting activity of TGF-.beta. on fibroblasts, (Postlethwaite, A. E. et al. (1987) J. Exp. Med. 165,251, modified according to Burk, R. (1973) PNAS 70,369), the inhibitory effect of TGF-.beta. on the growth of human A 375 melanoma cells (Brown, T. J. et al. (1987) J. Immunol. 139, 2977), inhibition of CCL-64 cell DNA synthesis assay (Graycar, J. L. etal., (1989) Molecular Endocrinology 3:1977-1986) or inhibition of the growth of a continuous mink lung epithelial cell line Mv-1-Lu (ATCC/CCL64) as described in the Examples hereinafter. Monomeric TGF-.beta.-like protein derived from any source or method can be folded into the corresponding dimeric, biologically active TGF-.beta.-like protein according to the present method. For example, the monomeric form of the TGF-.beta.-like protein can be derived from natural source or can be produced by means of recombinant DNA technology or synthetically by methods well known in the art. In the case the monomer is not suitable for in vitro folding due to contaminants, the solubilized and denatured monomer can be purified by chromatography, e.g. by size exclusion chromatography on e.g. Sephacryl S-100 HR. Before being folded, the monomeric TGF-.beta.-like protein has to be present in a denatured (i.e. unfolded), solubilized form. Capable of effectively denaturing and solubilizing proteins are so-called chaotropic agents well known in the art, which, in aqueous solution and in suitable concentrations, change the spatial configuration of the respective protein through alterations at the surface thereof, either through altering the state of hydration, the solvent environment, or the solvent-surface interaction. Examples of such chaotropic agents or denaturants include urea, guanidine hydrochloride, sodium thiocyanate at concentrations in the range of about 4 to about 9 M, and detergents such as SDS, which are supplied in concentrations in the order of 0.01 to 2 percent. Also, acidification of the aqueous solution containing the TGF-.beta.-like protein to a pH of about 2 to about 4, e.g. with a low molecular weight aliphatic organic acid, preferably having 2, 3 or 4 C-atoms, more preferably acetic acid, as well as basic conditions of e.g. pH 10 and above and elevated temperatures will result in denaturation and solubilization of the monomer. The monomer is then made subject to "folding conditions" which allow the recovery of the biologically active dimer. The term "folding conditions" refers to conditions under which intra and interchain disulfide bond formation is promoted and the denatured monomer is permitted to assume a conformation associated with the biological activity. This process does not involve any change in the primary structure (i.e. the amino acid sequence) of the monomer, but relates to the formation of the three-dimensional conformation of the dimeric product which is associated with the biological activity. This process includes the formation of disulfide bonds and the association of monomers into a dimeric, biologically active structure. For this purpose the denatured monomer is treated with a folding buffer which comprises (a) a mild detergent as defined hereinafter and (b) an organic solvent selected from the group consisting of DMSO, DMF, DMSO.sub.2 and any mixture of two or three of the group consisting of DMSO, DMSO.sub.2 and DMF, at a neutral or alkaline pH and at a reasonable temperature, e.g. between about 0.degree. C. and about 40.degree. C. A preferred pH is between about 7 and about 10, more preferred in the case of DMSO is about pH 9 to 9.5, in the case of DMF is about pH 8.5 and in the case of DMSO.sub.2 is about pH9.5. Conventional buffer systems which can be used for folding according to the present invention are buffers which provide sufficient buffer capacity between pH 6 and 10. All buffers that have no inhibiting effect on the folding of proteins are applicable in the present invention. For example, suitable buffers are Tris, bis-Tris or piperazine buffers. The buffers may contain additionally a salt, if desired, and a basic amino acid, if desired. Salts which can be used in the folding buffer are, for example, salts of Na.sup.+, Li.sup.+, K.sup.+, NH.sub.4.sup.+, Mg.sup.2+, Ca.sup.2+, or Mn.sup.2+ with Cl.sup.-, F.sup.-, Br.sup.-, J.sup.-, HCO.sub.3.sup.-, SO.sub.4.sup.2-, phosphate, acetate, cyanate or rhodanid, or other alkali metal--or alkaline earthmetal--halogen or pseudohalogen compounds at a concentration of up to 3 M. Preferred is NaCl at a concentration of 1 to 2 M. A basic amino acid which can be used in the folding buffer is, for example, arginine, preferably in a concentration of 0.5 M. The preferred concentration of DMSO, DMSO.sub.2 or DMF for the purpose of the present invention is from about 5% to about 40%, more preferably from about 10% to about 30%. The even more preferred concentration of DMSO is about 10% to about 30%, even more preferably about 20%; for DMF the even more preferred concentration is about 10% to about 30%, even more preferably about 10%; for DMSO.sub.2 the even more preferred concentration is about 10%. Mixtures of DMSO and DMF or of DMSO and DMSO.sub.2 or of DMF and DMSO.sub.2 can be used in a concentration of about 5% to about 40%, preferably of about 10% to about 30%, more preferably about 10% to about 20% for the solvents combined. A mild detergent suitable for the folding of a protein of the TGF-.beta. superfamily according to the present invention is any detergent which permits folding of the monomeric TGF-.beta.-like protein into the spatial conformation which after dimerization is associated with the biological activity, while retaining said monomer in a soluble form. Such detergents can be non-ionic, cationic, anionic or zwitterionic. Preferred detergents are the non-ionic detergent digitonin and, more preferred, the zwitterionic detergents 3-(3-chlolamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS) and 3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO). It is also possible to use a mixture of the detergents, e.g. a mixture of CHAPS and CHAPSO. Mild detergent is preferentially present in the folding buffer at a concentration of about 1 to 100 mM, more preferably at a concentration of 30 to 60 mM, even more preferably at a concentration of 30 mM. In a preferred embodiment of the present invention the folding buffer additionally contains a reducing substance. A suitable reducing substance which encourages the formation of disulfides in proteins or peptides is e.g. a low molecular weight sulfhydryl reagent selected from the group consisting of glutathione in its reduced form, dithiothreitol in its reduced form, .beta.-mercaptoethanol in its reduced form, mercaptomethanol in its reduced form, cysteine and cysteamine. However, the method also works if no such substance is present. A suitable concentration for the sulfhydryl reagent is e.g. about 1 to 100 mM, preferably about 1 to100 mM, more preferably about 2.5 mM The folding is performed at reasonable temperatures, for example between about 0 and about 40.degree. C., preferably at about 4.degree. C., and for a reasonable time period, for example between about 2 and about 720 h. Since the duration of the folding depends on the temperature used, the temperature may be optimized for any desired folding time period and vice versa. The production of a dimeric, biologically active TGF-.beta.-like protein according to the present invention may be performed in a one step procedure, wherein the monomer of said protein is transferred to the folding buffer and the reaction mixture is incubated for a time period of e.g. 2 hours up to 7 or more days at a temperature between e.g. 0.degree. C. and 40.degree. C., preferably 4.degree. C. while folding and dimerization continuously take place. The protein concentration during the folding reaction is of considerable importance since when being too high, the monomers might undergo substantial aggregation leading to the formation of undesired higher-order oligomers. Final yields of dimeric product are increased, if the protein concentration is less than about 2 mg/ml, a concentration range of 0.01 to 0.5 mg/ml is preferred. Preferred examples of folding experiments according to the present invention for the folding of TGF-.beta.3 are as follows: 0.1 mg/ml TGF-.beta.3, 100 mM Tris, optionally 1 to 50 mM of a substance selected from the group consisting of reduced glutathione, cysteine, cysteamin, and .beta.-mercaptoethanol (however, as already stated above, the method also works if no sylfhydryl redox system is present), 1 M NaCl, 0.5 M arginine, 20 to 30% DMSO, 30 mM CHAPS or CHAPSO, pH 9 to 9.5; or 0.1 mg/ml TGF-.beta.3, 100 mM Tris, 2.5 mM of a substance selected from the group consisting of reduced glutathione, cysteine, cysteamin, and .beta.-mercaptoethanol, (however, as already stated above, the method also works if no sylfhydryl redox system is present), 1 M NaCl, 0.5 M arginine, 10% DMF, 30 mM CHAPS or CHAPSO, pH 8.5. After folding, the biologically active dimer is purified in order to remove incompletely folded TGF-.beta.-like protein and impurities, in particular, pyrogens or other endotoxins which might be present in the preparation after production of the recombinant protein in microbial host cells. Separation of the dimer is performed by chromatography such as sizing gel chromatography, hydrophobic interaction chromatography or ion exchange chromatography, e.g. on a Mono S column and reverse phase HPLC. The present invention further relates to dimeric biologically active TGF-.beta.-like proteins when produced according to the process of the invention. These TGF-.beta.-like proteins can be used in a variety of therapeutic modalities. The following examples illustrate the invention without being meant to be limitative. |
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