OXIDATIVE RENATURATION OF HEN EGG-WHITE LYSOZYME - FOLDING VS AGGREGATION

Since the inception of recombinant DNA technology, different strategie s have been developed in the isolation, renaturation, and native disul fide bond formation of proteins produced as insoluble inclusion bodies in Escherichia coli. One of the major challenges in optimizing renatu ration processes is to prevent the formation of off-pathway inactive a nd aggregated species. On the basis of a simplified kinetic model desc ribing the competition between folding and aggregation, it was possibl e to analyze the effects of denaturant and thiol/disulfide concentrati ons on this competition. Although higher guanidinium chloride (GdmCl) concentrations resulted in higher renaturation yields, the folding rat e was negatively affected, indicating an optimum range of GdmCl for op timum renaturation rates and yields. Similarly, higher total glutathio ne concentrations resulted in higher yields but decreased rates, also indicating an optimum total glutathione concentration for optimum rena turation rates and yields (6-16 mM), with an optimum ratio of reduced to oxidized glutathione between 1 and 3. To characterize the nature of aggregates, aggregation experiments were performed under different ox idizing/reducing conditions. It is shown that hydrophobic interactions between partially folded polypeptide chains are the major cause of ag gregation. Aggregation is fast and aggregate concentration does not si gnificantly increase beyond the first minute of renaturation. Under co nditions which promote disulfide bonding, aggregate size, but not conc entration, may increase due to disulfide bond formation, resulting in covalently bonded aggregates.