Lysozyme Refolding Mediated By Dye-Ligand Affinity-Based Reverse Micelles

A novel protein refolding method using reverse micelles has been developed and the reports by now are very limited. Except for one report that used nonionic reverse micelles, all of the others used reverse micelles formulated with ionic surfactant, AOT, and none of them coupled affinity ligand. In this paper, a dye-ligand affinity-based reverse micellar system formulated with nonionic surfactant (RMS) was applied to the refolding of denatured/reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane.The refolding method and the operating conditions, such as concentrations of Span 85, CB, urea, GSSG, GSH and lysozyme,pH,W₀ and the time, were optimized. Under the best conditions, complete renaturation of lysozyme at 3 to 3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous under the same conditions gave much lower activity recovery, and the present system is advantageous over nonionic reverse micelles in protein refolding. Moreover, Over 95% of the refolded lysozyme was recovered from CB/Span 85/n-hexane reverse micelles by a stripping solution of 0.5 mol/L MgCl2 and the secondary structure of the refolded lysozyme was found the same as the native lysozyme. Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery. Co-solvent (Hexanol) and co-surfactant (Tween 85) were also successfully introduced to the system, and they could further give rise to protein refolding.Based on the excellent refolding result by RMS above, we used dye-ligand affinity-based reverse micelles and the artificial chaperone system (ACS) together to refold lysozyme. The method and important parameters, including the concentrations of urea, GSSG, GSH, lysozyme, CTAB, andβ-CD, and W₀, were comprehensively examined. And now the concentration of lysozyme that could be completely refolded rose to 4 mg/mL. Moreover, we combined other methods with RMS, such as additives, which also gave advantageous to lysozyme renaturation.