| Escherichia coli BL21 (DE3) (Invitrogen) was transformed with the plasmid. Bacteria were grown at 37 ?‹C overnight, and isopropylthiogalactoside was added to a final concentration of 1 mM. Induction was continued at 30 ?‹C for 4 h, and bacteria were harvested by centrifugation at 3,000 x g, and the cell pellet was suspended in 30 ml of lysis buffer (20 mM sodium phosphate, pH 7.8, containing 0.5 M NaCl) containing 1 tablet of complete EDTA free protease inhibitor cocktail (Roche Molecular Biochemicals). After sonication, the suspension was centrifuged at 4,000 ?~ g for 30 min at 4 ?‹C. The soluble fraction was bound to the Ni-NTA agarose (Qiagen) at 4 ?‹C overnight. The agarose was loaded to the column and washed with 5 bed volumes of wash buffer (20 mM sodium phosphate, pH 6.0, containing 0.5 M NaCl) three times. The column was subsequently washed with a solution composed of 6 volumes of isopropyl alcohol to remove bacterial endotoxins and 5 volumes of wash buffer containing 10 mM imidazole, and the protein was eluted with 3 ml of wash buffer containing stepwise concentration of 50 mM and 300 mM imidazole. Concentration of the eluted protein was determined by BCA protein assay kit (Pierce, Rockford, IL).
Refolding of recombinant protein was performed according to the method described by Tsumoto et al. Briefly, the eluted protein was diluted to a concentration of 7.5 uM with 50 mM Tris-HCl, pH 8.0, containing 0.2 M NaCl and 6 M guanidine hydrochloride and was reduced with 375 uM 2-mercaptoethanol (ƒÀ-ME) overnight. The eluted protein was subsequently dialyzed against 50 mM Tris-HCl, pH 8.0, containing 0.2 M NaCl and stepwise reducing concentration of 6, 3, and 2 M guanidine hydrochloride, and then against 50 mM Tris-HCl, pH 8.0, containing 0.2 M NaCl, stepwise reducing concentration of 1 and 0.5 M guanidine hydrochloride, 375 uM of oxidized glutathione (GSSG) and 0.4 M L-arginine to allow refolding of the proteins. The folded protein was finally dialyzed against PBS three times and then centrifuged at 12,000 x g for 20 min at 4 ?‹C to remove unfolded or aggregated proteins. The supernatant was checked by SDS-PAGE under non-reducing conditions, and the concentration of recombinant trappin was determined by BCA protein assay kit (Pierce).
|