Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron.
The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.
Ramotar
K
K
Samuel Lunenfield Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
Boyd
B
B
Tyrrell
G
G
Gariepy
J
J
Lingwood
C
C
Brunton
J
J
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D022622:Shiga Toxin 1
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D003603:Cytotoxins; D046911:Macromolecular Substances; D022622:Shiga Toxin 1