Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-02-18
2003-07-22
Romeo, David S. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S252330, C530S350000, C536S023500, C524S099000
Reexamination Certificate
active
06596511
ABSTRACT:
The present invention relates to methods of refolding proteins, particularly protein members of the transforming growth factor beta (“TGF-&bgr;”) 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.
BACKGROUND OF THE INVENTION
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. For example, Wang et al. in U.S. Pat. No. 5,013,649, incorporated herein by reference, describe the DNA sequences encoding bovine and human bone morphogenetic proteins 2A (now bone morphogenetic protein-2) and 2B (now bone morphogenetic protein 4), the corresponding proteins encoded by those DNA sequences, and processes for recombinant production of the BMP-2A (now BMP-2) and BMP-2B (now BMP4) proteins. These proteins are expected to have broad medical applicability in treatment of bone and cartilage injuries and disorders in mammals. In order to fulfill the expected medical need for these bone morphogenetic proteins, large quantities of biologically active protein will be needed.
Recombinant production of the bone morphogenetic proteins is possible both in eukaryotic and prokaryotic cell culture systems. A common occurrence in recombinant production of heterologous proteins in prokaryotic cells, such as bacteria, is the formation of insoluble intracellular precipitates known as inclusion bodies. While the bacteria are generally able to transcribe and to translate DNA sequences encoding heterologous proteins correctly, these prokaryotic cells are unable to fold some heterologous proteins sufficiently correctly to allow for their production in a soluble form. This is particularly true of prokaryotic expression of proteins of eukaryotic origin, such as the bone morphogenetic proteins. Formation of incorrectly folded heterologous proteins has to some extent limited the commercial utility of bacterial fermentation to produce recombinant mammalian proteins. When produced in bacteria, the recombinant bone morphogenetic proteins are often similarly found in inclusion bodies in an aggregated, biologically inactive form.
Several methods for obtaining correctly folded heterologous proteins from bacterial inclusion bodies are known. These methods generally involve solubilizing the protein from the inclusion bodies by denaturing the protein using acids or a chaotropic agent. Subsequently, protein is diluted into a refolding buffer that supports renaturation to a biologically active form. When cysteine residues are present in the primary amino acid sequence of the protein, it is often necessary to accomplish the refolding in an environment which allows correct formation of disulfide bonds (a redox system). General methods of refolding are disclosed in Kohno,
Meth. Enzym
., 185:187-195 (1990).
EP433225 describes a method for refolding transforming growth factor &bgr; (TGF-&bgr;)-like proteins which employs, in addition to a chaotropic agent and a redox system, a solubilizing agent in the form of a detergent. EP433225 predicts that the methods disclosed therein are generally applicable for refolding “TGF-&bgr;-like proteins”, based on the degree of homology between members of the TGF-&bgr; family. However, 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. In addition, the methods disclosed employ 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and other expensive compounds as the solubilizing agent.
Non-detergent sulfobetaines have been used in attempts to renature hen egg white lysozyme (HEWL) and &bgr;-D-galactosidase. However, these attempts have not been very effective and have practical drawbacks, such as yield. For example, certain sulfobetaines reduced the yield of &bgr;-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-&bgr; proteins.
SUMMARY OF THE INVENTION
It has been found, unexpectedly, that the dimeric proteins of the TGF-&bgr; superfamily, and particularly, bone morphogenetic proteins (BMPs), 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. Among the compounds useful in the present invention are members of the non-detergent zwitterions, pyridines, pyrroles and acid substituted aminocyclohexanes.
Accordingly, in one embodiment, the invention comprises methods of producing properly refolded proteins of the TGF-&bgr; 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-&bgr; 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 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. 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. For example, pyrrole-2 carboxylic acid, the pyrrole analog of nicotinic acid, is effective in the methods of the present invention. 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.
Unlike pyridines and pyrroles, 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. For example, 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. For example, 3-1-PPS is commercially available from Fluka Chemical Company, while Vitamin B is widely manufactured as a dietary supplement and food additive.
DETAILED DESCRIPTION OF THE INVENTION
Among the proteins which may be produced recombinantly using the methods of the present invention ar
Fitzpatrick ,Cella, Harper & Scinto
Genetics Institute LLC
Romeo David S.
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