Efficient Protein Refolding Using Surfactants at High Final Protein Concentration

Abstract

The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high final re- folded HEWL concentration (1 mg/mL). Hexadecyltrimethylammonium bromide (CTAB) and sucrose fatty acid monoester (DK-SS) were used to dissolve denatured HEWL without denaturants such as guanidine hydrochlo- ride (GuHCl) and urea. When denatured HEWL was perfectly dissolved in buffer solutions containing surfac- tants and dithiothreitol (DTT), the concentration of CTAB was about one-twentieth times less than that of DK-SS. The concentration of CTAB strongly affected the refolding yield, and the maximum refolding yield was obtained at 0.88 mM CTAB, which is around the critical micelle concentration of CTAB. The refolding yield was influenced by the molar ratio of oxidized glutathione (GSSG) to DTT, and the maximum refolding yield was ob- tained when [GSSG]/[DTT] was 1.5. The refolding yield was markedly dependent upon the solution pH of HEWL, and exhibited 80% at pH 5.2.