Methods for refolding proteins containing free cysteine residues
AssigneeBolder Biotechnology, Inc.
InventorsMary S. Rosendahl, George N. Cox, Daniel H. Doherty
Life of this patent
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Abstract
The present invention relates to novel methods for making and refolding insoluble or aggregated proteins having free cysteines in which a host cell expressing the protein is exposed to a cysteine blocking agent. The soluble, refolded proteins produced by the novel methods can then be modified to increase their effectiveness. Such modifications include attaching a PEG moiety to form PEGylated proteins.
Claims25
A method for preparing a refolded, soluble form of an insoluble or aggregated protein that is an anti-angiogenesis factor and which contains one or more added free cysteine residues, comprising the following steps, in order, of:
- a) causing a host cell to express a protein containing one or more added free cysteine residues that is an anti-angiogenesis factor in an insoluble or aggregated form;
- b) lysing the cell by chemical, enzymatic or physical means;
- c) separating soluble proteins from insoluble or aggregated proteins;
- d) following step (c) and prior to step (e), dissolving the insoluble or aggregated protein from step (c) in a solution comprising a denaturing agent, a reducing agent and a cysteine blocking agent that forms a reversible mixed disulfide with cysteine, to solubilize and reduce the insoluble or aggregated protein, and to expose the insoluble or aggregated protein to the cysteine blocking agent;
- and
- e) refolding the protein by reducing the concentrations of the denaturing agent and reducing agents to levels sufficient to allow the protein to renature into a soluble, biologically active form, wherein said refolding step occurs in the presence of the cysteine blocking agent, and wherein the cysteine blocking agent forms a reversible mixed disulfide with at least one added free cysteine residue in said protein.
A method for preparing and isolating a refolded, soluble form of an insoluble or aggregated protein that is an anti-angiogenesis factor and which contains one or more added free cysteine residues, comprising the steps of:
- a) causing a host cell to express a protein containing one or more added free cysteine residues that is an anti-angiogenesis factor in an insoluble or aggregated form;
- b) lysing the cell by chemical, enzymatic or physical means in the presence of a denaturing agent, a reducing agent and a cysteine blocking agent, wherein the insoluble or aggregated protein is denatured and reduced, and is in the presence of the cysteine blocking agent prior to step (c);
- c) refolding the protein by reducing the concentrations of the denaturing agent and reducing agents to levels sufficient to allow the protein to renature into a soluble, biologically active form, wherein said refolding step occurs in the presence of the cysteine blocking agent, and wherein the cysteine blocking agent forms a reversible mixed disulfide with at least one added free cysteine residue in said protein;
- and
- d) isolating the refolded, soluble protein from step (c).
Dependencies are inferred from the claim text.