Purification and characterization of toxin B from Clostridium difficile

Toxin B from Clostridium difficile was purified to homogeneity and characterized. Purification of toxin B was achieved by gel filtration, chromatography on two consecutive anion-exchange columns, and chromatography on a high-resolution anion-exchange column in the presence of 50 mM CaCl2. The molecular weight of toxin B was estimated to be 250,000 by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 500,000 by gel filtration. No subunits were apparent when the toxin was reduced and analyzed by SDS-PAGE. The estimated molecular weight of native toxin B indicated that dimers may form in solution. Toxin B was homogeneous by SDS-PAGE, native PAGE, and high-resolution anion-exchange chromatography. No secondary sequences were detected when the amino terminus of the toxin was sequenced, which also indicated that contaminating peptides were absent from the preparation. The amino terminus of toxin B was determined to be NH3-Trp-Leu-Val-Asn-Arg-Lys-Gln-Leu-Glu-Lys-Met-Ala-Asn-Val-ARg-Phe-Arg. One cytotoxic unit of toxin B was estimated to be 0.2 to 0.8 pg.