Role of Various Dilution Additives on the Refolding of Denatured-Reduced Lysozyme

The role of various dilution additives on the refolding of denatured-reduced chicken egg white lysozyme was investigated, and the effect of the concentrations of guanidinium chloride (GdmCl) and the additives was studied to find proper refolding conditions. It was found that the addition of arginine, acetamide, acetone, thiourea and glycerol was effective in improving the refolding yield. In addition, there was an optimal additive concentration for each additive, which made the refolding reach the highest yield. It was also found that the refolding-promoting mechanism of acetamide, arginine, acetone and thiourea was the same as that of GdmCl,i.e. GdmCl could be replaced by these additives as the refolding promoter. For example, at a GdmCl concentration of 0.2 mol/L, the presence of 2 mol/L acetamide improved the refolding yield to 90%, while at a low GdmCl concentration (0.06 mol/L), 4 mol/L acetamide was needed to yield 90% refolding. On the other hand, the effect of glycerol and GdmCl on protein refolding was additive. That is, increasing glycerol concentration at a proper GdmCl concentration could obtain a high refolding yield of protein.