Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
  • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
  • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• • C—CHEMISTRY; METALLURGY
  • 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
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/495—Transforming growth factor [TGF]
• • C—CHEMISTRY; METALLURGY
  • 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
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor

Definitions

• the present invention relates to methods of refolding proteins, particularly protein members of the transforming growth factor beta (“TGF- ⁇ ”) superfamily of proteins, such as the bone morphogenetic proteins (“BMPs”). These methods are particularly useful in improved processes for preparation of biologically active dimeric recombinant bone morphogenetic proteins produced in insoluble form from bacterial cell cultures.
• TGF- ⁇ transforming growth factor beta
• BMPs bone morphogenetic proteins
• bone morphogenetic proteins A number of proteins referred to in the art as bone morphogenetic proteins have recently been identified which are able to induce bone or cartilage formation when implanted into mammals.
• BMP-2A now BMP-2A
• BMP-2B BMP-2B
• EP433225 describes a method for refolding transforming growth factor ⁇ (TGF- ⁇ )-like proteins which employs, in addition to a chaotropic agent and a redox system, a solubilizing agent in the form of a detergent.
• TGF- ⁇ transforming growth factor ⁇
• EP433225 predicts that the methods disclosed therein are generally applicable for refolding “TGF- ⁇ -like proteins”, based on the degree of homology between members of the TGF- ⁇ family.
• the present inventors have found that the methods disclosed in EP433225 produce undesirably low yields of correctly folded, biologically active dimeric protein when applied to numerous bacterially produced BMPs.
• the methods disclosed employ 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and other expensive compounds as the solubilizing agent.
• CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanes
• Non-detergent sulfobetaines have been used in attempts to renature hen egg white lysozyme (HBEWL) and ⁇ -D-galactosidase.
• HBEWL hen egg white lysozyme
• ⁇ -D-galactosidase ⁇ -D-galactosidase
• these attempts have not been very effective and have practical drawbacks, such as yield.
• certain sulfobetaines reduced the yield of ⁇ -D-galactosidase by a factor of 100-fold.
• Goldberg et al., Folding Design., 1:21-27 (1996) Accordingly, these molecules have not been shown to be broadly effective as refolding agents, particularly for use in refolding multimeric proteins such as TGF- ⁇ proteins.
• the dimeric proteins of the TGF- ⁇ superfamily can be efficiently produced and refolded from bacterial cultures, such as E. coli, using methods which employ as refolding agents non-detergent nitrogen-containing compounds for the renaturation and correct refolding of dimeric protein.
• BMPs bone morphogenetic proteins
• the compounds useful in the present invention are members of the non-detergent zwitterions, pyridines, pyrroles and acid substituted aminocyclohexanes.
• the invention comprises methods of producing properly refolded proteins of the TGF- ⁇ superfamily from bacterial cell cultures using a non-detergent compound as a reagent in the method.
• the bacterial cell culture is preferably E. coli, but may be another bacterial or prokaryotic cell culture type.
• the protein may be any protein from the TGF- ⁇ superfamily, and is preferably a member of the BMP family, or the growth and differentiation factors (“GDFs”), as well as MP52 and other proteins as described further herein.
• GDFs growth and differentiation factors
• the non-detergent compound may be nitrogen-containing and/or zwitterionic, and preferably includes an aromatic or aliphatic ring, is preferably nitrogen containing, and is preferably substituted with a substituent which includes an electrophilic or electron accepting end group, such as a carboxyl or sulfhydryl group.
• a substituent which includes an electrophilic or electron accepting end group, such as a carboxyl or sulfhydryl group.
• Other end groups which may be useful in the present invention include amide groups.
• the non-detergent compound is preferably selected from the group consisting of sulfobetaines, pyridines, pyrroles and aminocyclohexanes.
• the non-detergent zwitterions useful in the present methods include sulfobetaines and pyridinium propanesulfonates, such as 3-(1-pyridinio)-1-propanesulfonate (“3-1-PPS”).
• Pyridine compounds useful in the present invention are preferably acid or amide substituted, and include pyridine 3-sulfonic acid, pyridine-2 carboxylic acid [also known as nicotinic acid or niacin or Vitamin B], picolinic acid, 3-pyridylacetic acid hydrochloride, 4-pyridylacetic acid hydrochloride, isonicotinic acid and nicotinamide.
• Pyrrole compounds which are useful in the present invention include the pyrrole analog of the above pyridine compounds.
• pyrrole-2 carboxylic acid the pyrrole analog of nicotinic acid
• Other non-detergent zwitterionic compounds useful in the present invention are compounds with a nitrogen containing aromatic ring, further containing an electron accepting substituent group, such as N-methyl-N-piperidine propane sulfonic acid, trigonelline hydrochloride, and 1-carboxymethyl pyridinium chloride.
• acid substituted aminocyclohexane compounds which are useful in the present invention contain an aliphatic ring with an amine substituent with an electron accepting group, such as a carboxyl or sulfhydryl group.
• an amine substituent with an electron accepting group such as a carboxyl or sulfhydryl group.
• 2-aminocyclohexane carboxylic acid, 3-(cyclohexylamino)- 1-propanesulfonic acid (CAPS), 3-cylclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO) and 2-(cylcohexylamino)ethanesulfonic acid (CHES) are each effective in the methods of the present invention.
• the methods of the present invention are further advantageous in that many of these compounds are relatively inexpensive and commercially available.
• 3-1-PPS is commercially available from Fluka Chemical Company, while Vitamin B is widely manufactured as a dietary supplement and food additive.
• proteins which may be produced recombinantly using the methods of the present invention are: BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, disclosed for example in U.S. Pat. Nos. 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication WO91/18098; and BMWP-9, disclosed in PCT publication WO93/00432, BMP-10, disclosed in PCT application WO94/26893; BMP-11, disclosed in PCT application WO94/26892, BMP-12 or BMP-13, disclosed in PCT application WO 95/16035, or BMP-15, disclosed in U.S.
• TGF- ⁇ superfamily proteins of the TGF- ⁇ superfamily which may be produced by the methods of the present invention include Vgr-2, disclosed in Jones et al., Mol. Endocrinol., 6:1961-1968 (1992); BMP, disclosed in WO94/01557; HP00269, disclosed in JP Publication number: 7-250688; and MP-52, disclosed in PCT application WO93/16099, and any of the growth and differentiation factors [GDFs], including those described in PCT applications WO94/15965; WO94/14949; WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/018415; WO96/02559 and others.
• GDFs growth and differentiation factors
• the methods of the present invention may be used to produce commercial scale quantities of BMP homodimers or heterodimers from bacteria and refolded into biologically active dimeric molecules. Production of heterodimers of BMPs is described, for example, in WO93/19229. The disclosures of all of the above applications are hereby incorporated by reference.
• Any bacterial species may be used to generate recombinant BMP for refolding in the method of the invention.
• Bacillus subtilis, Pseudomonas or Escherichia coli is used to produce inclusion bodies containing BMP.
• Escherichia coli is used to produce inclusion bodies containing BMP for refolding in the method of the invention.
• Any strain of E. coli may be used to produce BMP for refolding in the method of the invention, so long as that strain is capable of expression of heterologous proteins.
• E. coli strain GI724 A.T.C.C. accession number 55151
• GI774 without thyA
• the methods of the present invention may be used to produce BMPs in bacteria using known methods. It may be necessary to modify the N-terminal sequences of the BMP in order to optimize bacterial expression. For example, because cleavage of the bond between formyl-methionine and glutamine is inefficient in E. coli, the N-terminus of the native mature BMP-2 protein (Met-gln-ala-lys) is modified by deletion of the glutamine residue to yield an N-terminus more suitable for BMP-2 production in E. coli (Met-ala-lys-his). Other bacterial species may require analogous modifications to optimize the yield of the mutant BMP obtained therefrom. Such modifications are well within the level of ordinary skill in the art.
• the modified or unmodified nucleotide sequence which encode BMPs may be inserted into a plasmid suitable for transformation and expression of those heterologous proteins in bacteria.
• Any bacterial expression plasmid may be used, so long as it is capable of directing the expression of a heterologous protein such as BMP in the bacteria chosen.
• Acceptable species of bacteria include B. subtilis, species of Pseudomonas, and E. coli.
• Suitable expression plasmids for each of these species are known in the art.
• a suitable vector is described in Taniguchi et al., PNAS:USA, 77:5230-5233 (1980).
• the bacterial expression plasmid may be transformed into a competent bacterial cell using known methods. Transformants are selected for growth on medium containing an appropriate drug when drug resistance is used as the selective pressure, or for growth on medium which is deficient in an appropriate nutrient when auxotrophy is used as the selective pressure. Expression of the heterologous protein may be optimized using known methods.
• the BMP thus obtained will be present in insoluble, refractile inclusion bodies which may be found in pellets of disrupted and centrifuged cells.
• the inclusion bodies thus obtained may be solubilized using a denaturant such as guanidine hydrochloride or by acidification with an acid such as acetic acid or formic acid. If solubilized using a denaturant, a reducing agent such as ⁇ -mercaptoethanol, glutathione, or dithiothreitol is added with the denaturant. If the protein is solubilized by acidification, it must be reduced prior to acidification.
• a denaturant such as guanidine hydrochloride
• an acid such as acetic acid or formic acid.
• a reducing agent such as ⁇ -mercaptoethanol, glutathione, or dithiothreitol
• the solubilized heterologous protein may be further purified using known chromatographic methods such as size exclusion chromatography or reverse phase high performance liquid chromatography.
• the solution containing the BMP may then be reduced in volume or vacuum desiccated to remove chromatography buffer and redissolved in medium.
• reduced soluble protein may be renatured by diluting into refolding medium.
• suitable media may include the following:
• Other media may be suitable for renaturation, including media containing low levels of the chaotrope (e.g., guanidine hydrochloride) or the salt of the acid (e.g. acetate) used to solubilize the BMP inclusion bodies.
• Refolding is typically conducted at a BMP concentration of 1 to 100 ⁇ g/ml protein. Higher concentrations of protein may be refolded in accordance with the invention, for example up to about 1 mg/ml, but precipitates or aggregates may be present above protein concentrations of 100 ⁇ g/ml and the yield of active BMP homodimer or heterodimer may be decreased accordingly.
• the above procedure is performed utilizing equal amounts of two plasmids, each containing a coding sequence for a distinct BMP (e.g., pALBP2, encoding BMP-2 and pALBPX encoding BMP-X, where X is a BMP other than BMP-2).
• the plasmids are cultured separately, and the resulting inclusion bodies are solubilized and refolded in accordance with the methods described herein.
• the refolded protein monomers are mixed together in equivalent ratios and treated as described in the paragraph above.
• the resulting dimeric proteins are observed to include homodimers of BMP-2, as well as heterodimers of BMP-2/X. These species may be separated out from each other through procedures known in the art.
• the following conditions and media may be used: about 10 mM to about 100 mM Tris or other suitable buffer, preferably about 50 mM Tris, about 0.1 to about 4.0 M NaCl or other suitable salt, preferably about 1.0 M NaCl, about 0.05 to about 2.0 M refolding agent [non-detergent zwitterion, sulfobetaine, pyridine, pyrrole, or aminocyclohexane], preferably about 0.7M refolding agent, about 1 mM to about 10 mM EDTA or other suitable metal ion chelating reagent, preferably about 5 mM EDTA, a suitable redox system, such as glutathione, preferably at a ratio of about 1:10 to about 10:1 reductant to oxidant, at pH of about 7 to about 11, preferably about 8.5.
• a suitable redox system such as glutathione, preferably at a ratio of about 1:10 to about 10:1 reductant to oxidant,
• BMPs are disulfide bonded dimers in their active state, it is useful to include a redox system which allows formation of thiol/disulfide bonds in the method of the invention.
• redox systems are known.
• glutathione, dithiothreitol, ⁇ -mercaptoethanol, ⁇ -mercaptomethanol, cystine and cystamine may be used as redox systems at ratios of reductant to oxidant of about 1:10 to about 10:1.
• the ratio of reduced glutathione to oxidized glutathione is preferably 1 to 10; more preferably 1 to 1; and most preferably 2 to 1 of reduced form to oxidized form.
• the method of the invention may employ a salt moiety.
• the salt moiety is preferably NaCl, preferably at a concentration of about 0.1M to about 2.0M, preferably about 1.0M. It may be preferable to vary the sodium chloride concentration as the concentration of refolding agent varies.
• the pH of the refolding reaction of the present invention is preferably from about 7 to about 11; more preferably from about 8 to about 10 and most preferably about 8.5.
• the refolding reaction of the invention is performed at a temperature range from about 4° C. to about 37° C. More preferably, the refolding reaction is performed at 20° C.
• the refolding reactions of the present invention are allowed to proceed to completion between 8 and 120 hours and most preferably 96 hours.
• the extent of refolding of bone morphogenetic proteins obtained is monitored by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAISE) under non-reduced and reduced conditions.
• SDS-PAISE sodium dodecyl sulfate-polyacrylamide electrophoresis
• the BMP-4 homodimer will appear as a band of about 30 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; and the BMP-4 monomer appears as a band of about 13 kD under reduced conditions.
• the BMP-2/5 heterodimer will appear as a band of about 35 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; the BMP-2 monomer appears as a band of about 13 kD under reduced conditions; and the BMP-5 monomer appears as a band of about 15 kD under reduced conditions.
• the BMP-2/6 heterodimer will appear as a band of about 35 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; the BMP-2 monomer appears as a band of about 13 kD under reduced conditions; and the BMP-6 monomer appears as a band of about 15 kD under reduced conditions.
• the BMP-2/7 heterodimer will appear as a band of about 35 kD under non-reduced conditions on a 16 percent SDS-polyacrylamide gel; the BMP-2 monomer appears as a band of about 13 kD under reduced conditions; and the BMP-7 monomer appears as a band of about 15 kD under reduced conditions.
• a unifying concept for the compounds which are useful in the present invention appears to be molecules which contain a combination of two domains, usually nitrogen-containing molecules.
• the molecule should contain a hydrophobic domain, for example, an aromatic ring such as a pyridine or pyrrole ring.
• it may be a non-aromatic ring such as cyclohexane or a non-aromatic nitrogen containing ring in which nitrogen is in the form of a quaternary amine, such as N-methyl-N-piperidine propane sulfonic acid (also known as 1-methyl-1-sulfonylpropyl piperidine).
• the molecule should contain a substituent domain(s) which provides either zwitterionic or anionic attributes to the molecule.
• the substituent domain renders the molecule zwitterionic.
• the hydrophobic and substituent domains of the molecule should preferably be separated so as to present distinct regions.
• the electron accepting end group is no more than four carbons removed from the substituted aromatic or aliphatic nitrogen containing ring, more preferably no more than three carbons removed from the substituted aromatic or aliphatic nitrogen containing ring.
• the compounds useful as refolding agents in the present methods include non-detergent zwitterionic compounds.
• certain compounds may be “zwitterionic” at selected pH ranges, but not at others.
• Such compounds are preferably useful in the present invention at a pH in which the compound is zwitterionic.
• Preferred compounds in this group include non-detergent zwitterions, such as sulfobetaines, including certain pyridines and pyrroles.
• Substituted aliphatic nitrogen containing rings may also be useful when nitrogen is in the form of a quaternary amine.
• non-detergent sulfobetaine zwitterion 3-(1-pyridinio)-1-propanesulfonate (“3-1-PPS”), which is commercially available from Fluka Chemical Company, is useful in the present invention.
• compounds comprised of an aliphatic ring such as cyclohexane substituted with an amine group and an electron accepting substituent such as carboxyl or sulfhydryl group are also effective.
• This class of compounds may also be referred to as acid substituted aminocyclohexanes. Included in this group of compounds are some common biological buffers, or “Good's buffers”, including 2-(cyclohexylamino)ethanesulfonic acid (CHES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO).
• Another example of a preferred acid substituted aminocyclohexane useful in the present invention is 2-aminocyclohexane carboxylic acid.
• compounds useful in the present methods include compounds with substituted aromatic nitrogen containing rings such as pyridines and pyrroles substituted with electron accepting groups, preferably acid groups such as carboxyl or sulfhydryl groups, or amide groups.
• the pyridine nicotinic acid, or vitamin B is useful in the present invention.
• Other heterocyclic compounds for example purines, diazines, pyrazoles and imidazoles substituted with electron accepting substituerats, preferably acid groups such as carboxyl or sulfhydryl groups, or amide groups, may also be useful in the present invention, as well as derivatives of each of these compounds.
• the invention comprises methods of expressing properly refolded proteins of the TGF- ⁇ superfamily from bacterial cell cultures using one of the above refolding compounds, as described more fully below, as a reagent in the method.
• the bacterial cell culture is preferably E. coli, but may be another bacterial or prokaryotic cell culture type.
• the protein may be any protein from the TGF- ⁇ superfamily, and is preferably a member of the BMP family, or the growth and differentiation factors (“GDFs”), as well as MP52 and other proteins as described further herein.
• the refolding compound is preferably selected from the group consisting of non-detergent zwitterions, including non-detergent sulfobetaines, substituted pyridines, substituted pyrroles and acid substituted aminocyclohexanes.
• the refolding compound preferably comprises a hydrophobic domain, for example, an aromatic or aliphatic ring, and preferably further comprises a substituent domain, for example, an electron accepting end group, preferably an acid group such as a carboxyl or sulfhydryl group.
• substituent domains which may be useful in the present invention include amide groups.
• the non-detergent sulfobetaines useful in the present methods include pyridinium propanesulfonates, such as 3-(1-pyridinio)-1-propanesulfonate (“3-1-PPS”).
• Substituted pyridine compounds useful in the present invention include pyridine 3-sulfonic acid, pyridine-2 carboxylic acid [also known as nicotinic acid, niacin or Vitamin B], picolinic acid, 3-pyridylacetic acid hydrochloride, 4-pyridylacetic acid hydrochloride, isonicotinic acid and nicotinamide.
• Substituted pyrrole compounds which are useful in the present invention include the pyrrole analog of the above pyridine compounds.
• pyrrole-2 carboxylic acid pyrrole 3-sulfonic acid, 3-pyrrole acetic acid hydrochloride, 2-pyrrole acetic acid hydrochloride 2-pyrrole ethane sulfonic acid, 3-pyrrolehydroxymethane sulfonic acid.
• substituted aminocyclohexanes which are useful in the present invention are 2-aminocyclohexanecarboxylic acid, and Good's Buffers, such as CHES, CAPS and CAPSO. Illustrated below are some compounds which are useful in the present invention.
• substituted aminocyclohexanes which are useful in the present invention contain an aliphatic ring with an amine substituent with an electron accepting end group, such as a carboxyl or sulfhydryl group.
• an amine substituent with an electron accepting end group such as a carboxyl or sulfhydryl group.
• CHES, CAPS and CAPSO are each effective in the methods of the present invention. Illustrated below are various compounds which are useful.
• the in vitro biological activity of the refolded bone morphogenetic proteins may be monitored by the W-20 assay as set forth in the examples.
• Use of the W-20-17 bone marrow stromal cells as an indicator cell line is based upon the conversion of these cells to osteoblast-like cells after treatment with BMP [Thies et al., Journal of Bone and Mineral Research 5(2):305 (1990); and Thies et al., Endocrinology 130:1318-1324 (1992)].
• W-20-17 cells are a clonal bone marrow stromal cell line derived from adult mice by researchers in the laboratory of Dr. D. Nathan, Children's Hospital, Boston, Mass.
• Treatment of W-20-17 cells with BMP results in (1) increased alkaline phosphatase production, (2) induction of parathyroid hormone stimulated cAMP, and (3) induction of osteocalcin synthesis by the cells. While (1) and (2) represent characteristics associated with the osteoblast phenotype, the ability to synthesize osteocalcin is a phenotypic property only displayed by mature osteoblasts. Furthermore, to date the conversion of W-20-17 stromal cells to osteoblast-like cells has been observed only upon treatment with bone morphogenetic proteins.
• Nucleotides 1-2060 contain DNA sequences originating from the plasmid pUC-18 [Norrander et al., Gene 26:101-106 (1983)] including sequences containing the gene for ⁇ -lactamase which confers resistance to the antibiotic ampicillin in host E. coli strains, and a colE1-derived origin of replication.
• Nucleotides 2061-2221 contain DNA 5 sequences for the major leftward promotor (pL) of bacteriophage ⁇ [Sanger et al., J. Mol. Biol.
• Nucleotides 2222-2723 contain a strong 20 ribosome binding sequence included on a sequence derived from nucleotides 35472 to 35566 and 38137 to 38361 from bacteriophage lambda as described in Sanger et al., J. Mol. Biol. 162:729-773 (1982).
• Nucleotides 2724-3133 contain a DNA sequence encoding mature BMP-2 protein with an additional 62 nucleotides of 3′-untranslated sequence.
• Nucleotides 3134-3149 provide a “Linker” DNA sequence containing restriction endonuclease sites.
• Nucleotides 3150-3218 provide a transcription termination sequence based on that of the E. coli asp A gene [Takagi et al., Nucl. Acids Res. 13:2063-2074 (1985)].
• Nucleotides 3219-3623 are DNA sequences derived from pUC-18.
• Plasmid pALBP2-781 was transformed into the E. coli host strain GI724 (F, lacI q , lacp L8 , ampC:: ⁇ cI + ) by the procedure of Dagert and Ehrlich, Gene 6:23 (1979). Transformants were selected on 1.5% w/v agar plates containing IMC medium, which is composed of M9 medium [Miller, “Experiments in Molecular Genetics,” Cold Spring Harbor Laboratory, New York (1972)] supplemented with 1 mM MgSO 4 , 0.5% w/v glucose, 0.2% w/v casamino acids and 100 ⁇ g/ml ampicillin.
• GI724 transformed with pALBP2-781 was grown at 32° C. to an A 600 of 15 in IMC medium with 3 ⁇ MgSO 4 and 200 ⁇ g/ml ampicillin. For growth with other lines of E. coli which do not carry the thyA gene, ampicillin is not required in the medium.
• the glucose concentration of the culture was maintained at approximately 0.2% (wt./vol.).
• the pH was maintained at 7.2 with 7.5 M ammonium hydroxide. Tryptophan was added to a final concentration of 100 ⁇ g/ml and the culture was incubated for a further 4 hours at 37° C. During this time, BMP protein accumulated to approximately 10% of the total cell protein; all in the inclusion body fraction.
• BMP-2 eluting between 18 and 20 minutes was pooled and protein concentration was determined by A280 versus 0.5% (v/v) TFA, 17% 2-propyl alcohol, and 34% acetonitrile using the theoretical extinction coefficient based upon the amino acid content.
• BMP-2 pool was frozen at ⁇ 80° C. and subsequently lyophilized prior to refolding.
• Reversed-phase purified and lyophilized BMP-2 was solubilized as a 10 ⁇ concentrate at 250-500 ⁇ g/ml in 20 mM reduced glutathione. Soluble protein was immediately diluted into refolding buffer producing a refolding solution containing 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2mM reduced glutathione, 0.05-1.7 M 3-(1-Pyridinio)-1-propanesulfonate) and 25-50 ⁇ g/ml BMP-2 at pH 8.5. Refolding proceeded for three to four days at room temperature (approximately 20° C.).
• Refolding of E. coli produced BMP-2 was analyzed under non-reducing conditions using sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE). Sample aliquots were run on 10-20% acrylamide gels (Integrated Separation Systems) for approximately 2.5 hours at 75 milliamps. Protein was subsequently detected by silver stain. Refolding was scored as positive when the glutathione solubilized BMP-2 starting material appeared as a monomer of appropriate molecular weight and BMP-2 incubated in refolding media appeared as a dimer of the appropriate molecular weight under non-reducing conditions. BMP-2 dimer was also analyzed under reducing conditions by SDS-PAGE.
• BMP-2 appeared as a monomer of appropriate molecular weight. Biological activity of the refolded BMP-2 was tested using the assays described in Example 11 below. BMP-2 dimer was formed at 0.05 to 1.7 M 3-(1-Pyridinio)-1-propanesulfonate); optimally at 1.0 to 1.7 M 3-(1-Pyridinio)-1-propanesulfonate).
• Reversed-phase purified and lyophilized BMP-2 was solubilized as a 10 ⁇ concentrate at 500 ⁇ g/ml in 20 mM reduced glutathione. Soluble protein was immediately diluted into refolding buffer producing a refolding solution containing, 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 0.25-1.0 M nicotinic acid, and 50 ⁇ g/ml BMP-2 at pH 8.5 (pH was adjusted to approximately 8.5 with 5-10 M NaOH prior to dilution of BMP-2 concentrate). The resulting solution was held at room temperature (approximately 20° C.) for three to four days.
• BMP-2 dimer formation was observed at 0.25 to 1.0 M nicotinic acid.
• Recovery of BMP-2 dimer was optimal at 1.0 M nicotinic acid.
• Reversed-phase purified and lyophilized BMP-2 was solubilized as described in Example 4. Soluble protein was immediately diluted into refolding buffer producing a refolding solution containing 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 0.25-0.60 M pyridine-3 sulfonic acid, and 50 ⁇ g/ml BMP-2 at pH 8.5 (pH was adjusted to approximately 8.5 with 5-10 M NaOH prior to addition of protein concentrate). The resulting solution was held at approximately 20° C. for three to four days.
• BMP-2 dimer was formed at 0.25 to 0.60 M pyridine-3 sulfonic acid. Conversion of monomeric BMP-2 to dimer was greatest at 0.4-0.6 M pyridine-3 sulfonic acid.
• Reversed-phase purified and lyophilized BMP-2 was solubilized as a 10 ⁇ concentrate at 250-500 ⁇ g/ml in 20 mM reduced glutathione. Soluble protein was rapidly diluted into refolding buffer. The final refolding solution contained 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 0.25-0.70 M 2-(cylcohexylamino)ethanesulfonic acid (CHES) and 25-50 ⁇ g/ml BMP-2 at pH 8.3. Refolding proceeded for three to four days at room temperature.
• BMP-2 dimer was formed at 0.25 to 0.70 M CHES. Conversion of monomeric BMP-2 to dimer was greatest at 0.70 M CHES.
• Reversed-phase purified and lyophilized BMP-2 was solubilized as a 10 ⁇ concentrate at 500 ⁇ g/ml in 20 mM reduced glutathione. Soluble protein was diluted into refolding buffer producing a refolding solution containing 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 0.40-0.65 M pyrrole-2 carboxylic acid, and 50 ⁇ g/ml BMP-2 at pH 8.4 (pH was adjusted to approximately 8.4 with 5-10 M NaOH prior to dilution of BMP-2 concentrate). The resulting solution was held at room temperature (approximately 20° C.) for three to four days.
• BMP-13 was expressed as described in example 1.
• TE8.3(100: 10) buffer 100 mM Tris-HCl pH 8.3, 10 mM Na 2 EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF].
• PMSF phenylmethylsulfonyl fluoride
• Cells were lysed by three passes through a MicrofluidizerTM [model #MCF 100 T].
• the lysate was diluted to approximately 120 ml with TE8.3 100:10 buffer.
• a pellet of inclusion body material was obtained by centrifugation at 15,000 ⁇ g.
• the supernatant was decanted, and the inclusion body material was suspended in 50 ml TE8.3(100:10) which also contained 1% Triton-X100.
• the resuspended inclusion bodies were centrifuged for 10 minutes at 15,000 ⁇ g, and the supernatant was decanted.
• the pellet was suspended in TE8.3(20:1) buffer (20 mM Tris-HCl pH 8.3, 1 mM Na 2 EDTA, 1 mM PMSF) which also contained 1% dithiothrietol [DTT].
• TE8.3(20:1) buffer (20 mM Tris-HCl pH 8.3, 1 mM Na 2 EDTA, 1 mM PMSF
• DTT dithiothrietol
• Sepharose S-100TM size exclusion column (83 cm ⁇ 2.6 cm; ⁇ 440 ml bed) in 20 ml increments.
• the Sepharose S-100TM column was run with a mobile phase of 1% acetic acid at a flow rate of 1.4 ml/min. Fractions corresponding to BMP-13 monomer were detected by absorbance at 280 nm. Protein concentration was determined using a computer calculated extinction coefficient and molecular weight after measuring the absorbance of the pool at 280 nanometers. This size exclusion column pooled material was used as starting material for refolding reactions.
• cells were lysed as above, but the initial inclusion body material pellet was dissolved in 8 M guanidine-HCl, TE8.5(100:10) buffer (100 mM Tris-HCl pH 8.5, 10 mM Na 2 EDTA which contained 100 mM DTT, and incubated at 37° C. for 1 hour. This material was centrifuged at 12,000 ⁇ g for 15 minutes at room temperature.
• BMP-13 protein in 1% acetic acid or in reverse phase buffer containing 0.1% TFA, 30-40% acetonitrile was dried or reduced in volume using a speed vacuum, redissolved as a concentrate with a few microliters of purified water or reduced glutathione, and allowed to dissolve completely for 5 to 10 minutes. Soluble protein was subsequently diluted into refolding buffer.
• the final refolding solution contained 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 1.0 M 3-(1-Pyridinio)-1-propanesulfonate) and 50 ⁇ g/ml BMP-13 at pH 8.4.
• Refolding proceeded for three to four days at room temperature (20-23° C.). Refolding of the E. coli produced BMP-13 in 3-(1-Pyridinio)-1-propanesulfonate) was analyzed under reducing and non-reducing conditions using 16% Tricine-sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) or 10-20% sodium dodecyl sulfate polyacrylamide electrophoresis. Protein was detected by silver stain. Refolding was scored as positive when the BMP-13 appeared as a dimer of the appropriate molecular weight under non-reducing conditions and as a monomer of appropriate molecular weight under reducing conditions. BMP-13 dimer was produced in the presence of 1.0 M 3-(1-Pyridinio)- 1 -propanesulfonate).
• Acetic acid solubilized and size exclusion purified BMP-13 was lyophilized and subsequently solubilized as a 10 ⁇ concentrate at 500 ⁇ g/ml in 20 mM reduced glutathione. Soluble protein was immediately diluted into refolding buffer producing a refolding solution containing 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 0.65-1.0 M nicotinic acid, and 50 ⁇ g/ml BMP-13 at pH 8.5 (pH was adjusted to approximately 8.5 with 5-10 M NaOH prior to dilution of BMP-13 concentrate). The resulting solution was held at room temperature (approximately 20° C.) for three to four days.
• BMP-2 and BMP-13 were expressed as described in Example 1.
• BMP-2 inclusion bodies were isolated, and BMP-2 was subsequently solubilized and purified as outlined in Example 2.
• Reduced BMP-13 monomer was prepared as described in Example 8. Lyophilized BMP-2 was redissolved as a concentrate in purified water.
• BMP-2 and BMP-13 were diluted into refolding buffer resulting in equal mass ratios in the refolding solution.
• the final final composition of the refolding solution was 50 mM TRIS, 5 mM EDTA, 1 M NaCl, 1 mM oxidized glutathione, 2 mM reduced glutathione, 1.0 M 3-(1-pyridinio)-1-propanesulfonate), 50 ⁇ g/ml BMP-2 and 50 ⁇ g/ml BMP-13 at approximately pH 8.5. Refolding proceeded for three to four days at room temperature (20-23° C.). Refolding of the E.
• BMP-2/13 produced BMP-2/13 in 3-(1-pyridinio)-1-propanesulfonate) was analyzed under reducing and non-reducing conditions using 16% Tricine-sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) or 10-20% sodium dodecyl sulfate polyacrylamide electrophoresis. Protein was detected by silver stain. Refolding was scored as positive when the BMP-2/13 appeared as a heteradimer of the appropriate molecular weight under non-reducing conditions and as monomeric proteins of appropriate molecular weight under reducing conditions. BMP-2/13 heterodimer was produced in the presence of 1.0 M 3-(1-pyridinio)-1-propanesulfonate.
• a desalting step may be added prior to assay for activity of refolded protein.
• W-20-17 cells are plated into 96 well tissue culture plates at a density of 10,000 cells per well in 200 ⁇ l of medium (DME with 10% heat inactivated fetal calf serum, 2 mM glutamine). The cells are allowed to attach overnight in a 95% air, 5% co 2 incubator at 37° C.
• medium DME with 10% heat inactivated fetal calf serum, 2 mM glutamine. The cells are allowed to attach overnight in a 95% air, 5% co 2 incubator at 37° C.
• test samples and standards are allowed a 24 hour incubation period with the W-20-17 indicator cells. After the 24 hours, plates are removed from the 37° C. incubator and the test media are removed from the cells.
• the W-20-17 cell layers are washed three times with 200 ⁇ l per well of calcium/magnesium free phosphate buffered saline and these washes are discarded.
• reaction is stopped by adding 100 ⁇ l of 0.2 N NaOH to each well and placing the assay plates on ice.

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