Effect of Glutathione Redox System on Lysozyme Refolding in Size Exclusion Chromatography

The refolding step is of key importance for industrially produced protein aggregates to regain their biological activities. A number of strategies, including the direct dilution and chromatographic methods, have been utilized to improve the performance of the refolding process; however, refinement and advances on these methods are ongoing. Using lysozymes as a model system, we presented a study on the effects of redox system GSH/GSSG on protein refolding process in size exclusion chromatography (SEC). Attempts were first made to understand how redox constituents of refolding buffer affected the direct dilution or SEC refolding performance, and then to compare the operating mechanisms between the direct dilution and SEC refolding methods. Our results showed that, the combination of reduced as well as oxidized forms of glutathione was able to provide a favourable environment for protein renaturation. The performance of denatured protein refolding could be optimized via an appropriate choice of the relative distribution between GSH and GSSG. Moreover, our research further indicated that SEC packing provided an inhibitory action against aggregation, especially at lower dilution factors (or higher protein concentrations). We believe that the outcome from this study may facilitate the development of a more effective strategy for protein refolding processes.