Nickel resin

Production of 6xHis-tagged wild-type and mutant CSB carrying amino acids from 2 to 322 was carried out essentially as described^{59 (link), 63 (link)} with minor modifications. Induction of CSB proteins was carried out overnight with 1 mM isopropylthiogalactoside at room temperature. The cell pellet was resuspended in Binding buffer [20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, 1 mM PMSF] and lysed by sonication. Triton X-100 was then added to 0.1% and the lysate was shaken at 4 °C for 30 min. Following centrifugation, the supernatant was incubated with nickel resin (Qiagen) at 4 °C for 2 h. The beads were washed once in Binding buffer, three times in Wash buffer [20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 50 mM imidazole, 10 mM β-mercaptoethanol and 1 mM PMSF] and then eluted three times with an elution buffer [20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 880 mM imidazole and 10 mM β-mercaptoethanol]. The elutions were combined and dialysed against a dialysis buffer [20 mM HEPES pH7.9, 500 mM NaCl, 20% glycerol, 3 mM MgCl₂ and 1 mM DTT]. For cyclin A/CDK2 kinase assays, 2.5 μg of His-tagged wild-type and mutant CSB fragments was incubated with or without 50 ng of active recombinant cyclin A/CDK2 (14–488, Millipore) in the presence of ATP according to the manufacturer’s protocol.

Transformed E. coli were grown in SOB medium and recombinant proteins expression was induced at OD₆₀₀ = 0.6, with 0.5 mM isopropyl-thio-β-D-galactoside at 37°C for 4 h. All 6xHIS-fused proteins were purified under native condition by affinity chromatography on nickel resin (Qiagen) and eluted with 250 mM imidazole (pH 8.0). Proteins were dialyzed against PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) using Slide-A-Lyzer dialysis cassettes (cut-off 3.5 kDa, Thermo Fisher Scientific, Rockford, IL, USA). The protein concentration was determined by Bradford assay (Thermo Fisher Scientific) and proteins were stored at -70°C until use.

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.

Purification of typhoid toxin was conducted as described previously (Song et al., 2013 (link)). Briefly, the genes encoding different versions of typhoid toxins in S. Typhi (as listed in the Key resources table) were cloned into the pET28a (Novagen) expression vector. E. coli strains carrying the plasmids encoding the different toxins 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 I, 0.1 mg/ml lysozyme, and 0.1% PMSF and lysed by passaging 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 (Song et al., 2013 (link)). Purified toxins were examined for purity on SDS-PAGE gels stained with coomassie blue.