Expression, and purification of recombinant TcdB proteins

Recombinant full-length TcdB proteins were expressed in Bacillus subtilis SL401 and purified as previously described [15]. In brief, expression was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 25°C for 16 hours. TcdB1^1-2098, TcdB1^544-2098, and TcdB1^1285-2366 were expressed in E. coli BL21 (DE3) and purified as His6-tagged proteins. E. coli cells were cultured at 37°C till OD₆₀₀ reached 0.8. Expression was induced with 0.5 mM IPTG at 16°C overnight. Proteins were purified by Ni-affinity chromatography and followed by Superdex-200 (GE Healthcare) size-exclusion chromatography.