title: Oxidative refolding from inclusion bodies.
abstract: This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.
authors.affiliation: Center for Structural Genomics of Infectious Diseases, Computational Institute, University of Chicago, Chicago, IL, 60557, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Nelson
authors.forename: Chung A
authors.initials: CA
authors.lastname: Lee
authors.forename: Daved H
authors.initials: DH
authors.lastname: Fremont
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry*; D008967:Molecular Biology / Q000379:methods*; D010084:Oxidation-Reduction; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D011506:Proteins