ABSTRACT
In the in vitro refolding of a well-characterized protein, the initial step is to unfold the molecule in the presence of denaturing agents; then renaturation is initiated on removal of denaturants. Refolding to the native protein is a spontaneous process. Proteins containing disulfide bonds are fully reduced by reagents that specifically cleave disulfides. The rechromatography identified intermediate species that ranged from partly folded molecules to the structure of the native protein. The restoration of native structure requires correct folding and pairing of half-cystine residues. The refolding process using size-exclusion high performance liquid chromatography (SEC). SEC is an important analytical tool to study the refolding of disulfide-containing proteins. The unfolded molecule has a large Stokes radius because it occupies a large volume. High-performance liquid chromatography separated intermediates because they were stabilized by disulfide bonds. No further changes in conformation were detected during rechromatography of the fractions; the elution time was the same on repeated chromatography.
