Method for refolding insoluble, misfolded insulin-like growth factor-I into an active conformation
AssigneeGenentech, Inc.
InventorsJudy Y. Chang, Nancy C. McFarland, James R. Swartz
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Abstract
A method for refolding insoluble, improperly folded IGF-I is provided, wherein the IGF-I, precipitated from prokaryotic host cells, it concurrently solubilized, unfolded, and refolded into a biologically active conformation in a single buffer. The buffer contains reducing agent and chaotropic agent to solubilize the IGF-I at concentrations sufficiently low to allow solubilization and folding to occur. Also provided is a triple-protease deficient E. coli host suitable for use in the process.
Claims22
A process for reactivating insoluble misfolded IGF-I contained in prokaryotic host cells, which process comprises:
- (a) isolating said IGF-I;
- and
- (b) incubating said isolated IGF-I at a concentration of about 0.5-5.5 mg IGF-I per ml in an alkaline buffered solution comprising 1-3 M chaotropic agent and the minimal concentration of reducing agent sufficient for solubilization, under conditions of incubation temperature and time whereby solubilization, unfolding, and refolding of the IGF-I all occur during the incubation.
A process for reactivating insoluble, misfolded IGF-I contained in prokaryotic host cells, which process comprises:
- (a) isolating said IGF-I;
- and
- (b) incubating said isolated IGF-I at a concentration of about 0.5-5.5 mg IGF-I per ml in an alkaline buffered solution comprising 1-3 M chaotropic agent and the minimal concentration of dithiothreitol sufficient for solubilization, under conditions of incubation temperature and time whereby solubilization, unfolding, and refolding of the IGF-I all occur during the incubation.
Dependencies are inferred from the claim text.