Expression and purification of recombinant TcdB. pHis-TcdB plasmid was transformed to B. megaterium protoplasts. Several transformed colonies were picked up and the expression of rTcdB was induced by xylose. (A) His-tag affinity purification of total lysate from B. megaterium. Lane 1: total bacterial lysate; Lane2: flow-through; Lane 3: wash; lane 4–6: elution fraction 1–3. (B) Anion-exchanging column fractionation after Ni-affinity chromatography. nTcdB: purified native TcdB from C. difficile culture supernatant. Lane 1: Elution fraction 2 from (A); Lane 2–8: fractions from a gradient salt elution. Fraction 5 and 6 contain purified rTcdB. (C) Western blot results of purified native TcdB and rTcdB of combined fraction 5 and 6 from (B). (D) Coomassie staining of SDS-PAGE from total sonication lysates a pHis-TcdB transformed clone (lane 1), concentrated supernatant from pHis-SP-TcdB transformed clone (lane 2) and purified native TcdB (lane 3). M indicates molecular weight marker and 250 kDa is showed.