Purification and biological properties of Escherichia coli verocytotoxin

• Published in: FEMS microbiology letters 1987, Vol.41 (1), p.63-68
• Main Authors: Petric, M., Karmali, M.A., Richardson, Susan, Cheung, Rose
• Format: Article
• Language: English
• Subjects: Cytotoxin; cytotoxins; Enterotoxin; Escherichia coli; Vero cell purification; Verocytotoxin
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.1987.tb02142.x

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 proteindeadenylylation. Carbon‐limited chemostat cultures showed low glutamine synthetase activity levels but the synthesis of the enzyme was derepressed when the cultures became N‐limited. 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.