Analysis of refolding variables to identify the optimal refolding conditions for MB109. (A) Top panel is non-reduced SDS-PAGE gel image showing the result of refolding in 0–4 M NaCl. Black and gray arrows indicate the refolded functional and chemical dimers, respectively. Middle panel shows the detail of each refolding condition. Bottom panel is densitometry of the functional dimer bands in the top SDS-PAGE image. The data show relative refolding yield in each tested condition as compared to the highest one (set as 100%). (B-F) Refolding of MB109 at pH 7–10 (B), in 0–4% CHAPS detergent (C), in different molar ratios of reduced (GSH) and oxidized (GSSG) glutathiones (D), at 0.05–0.4 mg/mL protein concentrations (E), and the time course of MB109 Refolding at 4°C (F). (G) Effect of secondary additives on the refolding of MB109. The background conditions were 1.5 mM NaCl, pH 8.3, 2% CHAPS, 1/1 mM GSH/GSSG, 0.2 mg/mL protein, 2 mM EDTA and 4°C for 7 days.