Purification of Bacterial Cytotoxins

Purification of typhoid toxin and cytolethal distending toxin (CDT) was conducted as described previously [1 (link), 72 (link)]. Briefly, the genes encoding typhoid toxin in Salmonella Typhi (pltA/pltB/6xHis-cdtB) or CDT in Campylobacter jejuni (cdtA/cdtC/6xHis-cdtB) were cloned into the pET28a (Novagen) expression vector. Escherichia coli strains carrying the different plasmids were grown at 37°C in LB media to an OD₆₀₀ of ~0.6, toxin expression was induced by the addition of 0.5 mM IPTG, and cultures were further incubated at 25°C overnight. Bacterial cell pellets were resuspended in a buffer containing 15 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 mg/ml DNase, 0.1 mg/ml lysozyme, and 0.1% PMSF and lysed by passage through a cell disruptor (Constant Systems Ltd.). Toxins were then purified from bacterial cell lysates through affinity chromatography on a Nickel-resin (Qiagen), ion exchange, and gel filtration (Superdex 200) chromatography as previously described [1 (link), 72 (link)]. Purified toxins were examined for purity on SDS-PAGE gels stained with coomassie blue.