Study on Efficient Protein Refolding Using Surfactants at High Final Protein Concentration

The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high
final refolded 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 hydrochloride (GuHCl) and urea. When denatured HEWL was perfectly dissolved
in buffer solutions containing surfactants 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 obtained 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.