Purification and characterization of verocytotoxin 2

Abstract

Verocytotoxin 2 (VT2) was purified from Escherichia coli strain E32511 using, as starting material, cells harvested from a Penassay broth culture incubated for 6 h at 37°C in the presence of mitomycin C (0.2 µg/ml). A crude extract of VT2, obtained by polymyxin B treatment of cell pellets, was purified using differential ammonium sulphate precipitation, and sequential column chromatography. The purified toxin was estimated to have a pI of 6.5 by chromatofocusing and a molecular weight of 42 000 by gel filtration; it had a specific activity of 1.39 × 106 CD50 units/mg protein in Vero cells, and resolved as a major band of Mr 35 000 and another band of <14 000 which migrated with the buffer front on reducing SDS-polyacrylamide gel electrophoresis. The purified toxin was not neutralized by VT1 antisera and antisera prepared to this toxin in rabbits did not neutralize VT1, but completely neutralized the activity of the homologous toxin. Purified VT2, when administered intravenously, was lethal to rabbits with an LD50 of 30 µg/kg. Rabbits displayed symptoms of overt haemorrhagic caecitis and flaccid paralysis.