Methods of making proteins and peptides containing a single free cysteine
AssigneeBolder Biotechnology, Inc.
InventorsGeorge N. Cox, Mary S. Rosendahl
Life of this patent
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Abstract
Disclosed is a method for refolding a protein or peptide that does not contain essential disulfides and that contains at least one free cysteine residue. Also disclosed are polymer IFN-γ conjugates that have been created by the chemical coupling of polymers such as polyethylene glycol moieties to IFN-γ, particularly via a free cysteine in the protein. Also disclosed are analogs of bioactive peptides that may be used to create longer acting versions of the peptides, including analogs of glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomodulin.
Claims31
A method for refolding an insoluble protein or peptide that lacks essential disulfides and that comprises one free cysteine residue, comprising the following steps:
- a) causing a host cell to express a protein or peptide that lacks essential disulfides and that comprises at least one free cysteine residue in an insoluble or aggregated form;
- b) lysing the host cell;
- c) isolating the protein or peptide;
- d) denaturing and reducing the protein or peptide in a solution comprising both a denaturing agent and a reducing agent, wherein said reducing agent does not form a mixed disulfide with the free cysteine in the protein or peptide, and wherein said reducing agent does not inactivate a thiol-reactive polyethylene glycol (PEG) or does not interfere with modification of the protein by a thiol-reactive PEG reagent;
- e) refolding the protein by reducing the concentrations of the denaturing agent and reducing agents in the solution of (d) to levels sufficient to allow the protein or peptide to renature into a soluble, biologically active form;
- f) wherein steps (d) and (e) occur in the absence of a cysteine blocking agent.
Dependencies are inferred from the claim text.