Purification and characterization of an Escherichia coli Shiga-like toxin II variant

Abstract

A Shiga-like toxin II variant was purified to homogeneity from Escherichia coli TB1(pCG6), which contained the toxin genes cloned in multicopy plasmid pUC18. The purification scheme involved polymyxin B extraction of the toxin from bacterial cells, followed by differential (NH4)2SO4 precipitation, anion- and cation-exchange fast-protein liquid chromatography, and immunoaffinity exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified toxin revealed three protein bands that migrated with calculated molecular weights of 33,000, 27,500, and 7,500. These bands correspond to values for the A, A1, and B subunits, respectively, that would be expected on the basis of the nucleotide sequence and comparison with data for Shiga toxin and other Shiga-like toxins. Electrophoresis under nonreducing conditions resulted in disappearance of the 27,000-molecular-weight band. Western blot (immunoblot) analysis revealed three protein bands with molecular weights of 33,000, 27,500, and 7,500. The purified toxin induced typical signs of edema disease in pigs injected intravenously with doses as small as 3 ng/kg of body weight. The 50% cytotoxic doses for Vero, PK15, and Madin-Darby bovine and canine kidney cells were 0.5, 2.0, 8.0, and 8.0 pg, respectively. The 50% lethal dose of purified toxin for mice was 0.9 pg by the intraperitoneal route. Approximately 75 micrograms of purified toxin was required to induce a 1-ml/cm fluid response in rabbit ileal loops. Antiserum to the Shiga-like toxin II variant neutralized homologous toxin, Shiga-like toxin II, and Verotoxin 2 but not Shiga-like toxin I.