Studies of the hydrodynamic volume changes that occur during refolding of lysozyme using size-exclusion...

A size-exclusion chromatography-based refolding process (SEPROS) has successfully been used to renature lysozyme at high concentrations. This process is based on the different hydrodynamic characteristics of folded and unfolded proteins and their interaction with gel filtration media. In this paper we have quantified the changes in Stokes radius, hydrodynamic volume and partition coefficient that occur when lysozyme is refolded from urea in a size-exclusion column. In 8 M urea partially folded and unfolded lysozyme were resolved using Superdex 75 HR. These two species were present at approximately the same concentration. As the urea concentration was decreased the unfolded species gradually decreased until at 4 M urea only partially folded lysozyme remained, which continued to fold on further reduction of the urea concentration. Using these results the initial mechanism for size exclusion chromatography protein refolding has been confirmed.