Purification and biological properties ofEscherichia coliverocytotoxin

A simple method was developed for purifying Escherichia coli H 30 verocytotoxin (VT). The toxin, released from the cells by exposure to polymyxin B, was subjected to differential ammonium sulphate precipitation and sequential chromatography on hydroxylapatite, chromatofocussing, Cibachron blue, and Sephadex G-100 columns. The purified toxin, of 39 kDa by gel filtration and a pI of 6.72, resolved as a band which migrated at 32 kDa and another band, of less than 14 kDa, which migrated with the buffer front on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified preparation was relatively heat-stable, and had a specific activity of 3 × 109 CD50 units/mg protein in Vero cells, and LD50 values of 0.2, 9.0, and 40 μg protein/kg in rabbits, rats, and mice, respectively. Antiserum to the toxin specifically neutralized H 30 VT, Shiga toxin, and VT activity from some clinical isolates of VT+E. coli, but not that from a porcine oedema disease strain.