coli strain TOP10F′. After confirming the

sequence, the cloned DNA was extracted from the plasmid using restriction enzymes (EcoRI and HindIII) and then subcloned into the pBluescript II SK(+) vector (Stratagene, La Jolla, CA, USA) digested with the same enzymes. The expression plasmid for Stx2-His was named pBSK-Stx2(His). The expression plasmid of the attenuated mStx2-His was generated from pBSK-Stx2(His) by changing the glutamic acid at position 167 and the arginine at position 170 of the A subunit into glutamine and leucine, respectively, by site-directed mutagenesis using a QuikChange II Site-directed Mutagenesis Kit (Stratagene) and two primer sets: Stx2A(E167Q)-f and Stx2A(E167Q)-r; and Stx2A(E167Q + R170L)-f and Stx2A(E167Q + R170L)-r. All primer sequences used in this study are listed in Table 1 and the plasmid map for pBSK-Stx2(His) is shown in Figure 1. The pBSK-Stx2(His) plasmid was transformed Dasatinib cell line into E. coli strain MV1184 (ara, Δ(lac-proAB), rpsL, thi (φ80lacZΔM15), Δ(srl-recA)306::Tn10 (tetr)/F′[traD36, proAB+, lacIq, lacZΔM15]). Each transformant was cultured in Luria–Bertani broth containing 50 μg/mL (final concentration) ampicillin overnight at 37°C. Next, 3 mL of culture was inoculated Staurosporine price into 1 L of CAYE broth (2% casamino acids, 0.6% yeast extract, 0.25% NaCl, 0.871% K2HPO4 and 0.25% glucose) containing a 0.1% (v/v)

trace salt solution (5% MgSO4, 0.5% MnCl2 and 0.5% FeCl3), 50 μg/mL of ampicillin, and 90 μg/mL of lincomycin (Pfizer, New York, NY, USA) and cultured for 48 hr at 30°C. The cells were collected by centrifugation (7600 g, 20 min) and sonicated in PBS (pH 7.4). After centrifugation (15,000 g, 90 min), the supernatant was applied to a 2 mL column of TALON metal affinity resin (Clontech, Mountain View, CA, USA) equilibrated with PBS, and then acetylcholine bound Stx2-His (or mStx2-His) was eluted by PBS containing 0.15 M imidazole. To remove the contaminated products of crude Stx2-His preparation, hydroxyapatite (Bio-Rad, Hercules, CA, USA) chromatography was conducted. Prior to chromatography, each crude preparation was dialyzed against 10 mM sodium phosphate buffer (pH 7.0) containing 1 M NaCl to avoid

aggregation and then applied onto a hydroxyapatite column equilibrated with the same buffer. After collecting the unabsorbed fractions, the bound proteins were eluted with 0.4 M sodium phosphate buffer (pH 7.0). Unabsorbed Stx2-His was concentrated by applying it onto fresh TALON affinity resin and the final products were dialyzed in PBS. Throughout the purification process, insoluble proteins which were yielded during the dialyzing steps and storage period at −30°C were removed by centrifugation (15,000 g, 30 min). Protein concentrations were determined with DC protein assay reagent (Bio-Rad) using BSA as a standard. The toxicity of each Stx2-His and EHEC-derived Stx2 (Nacalai Tesque, Kyoto, Japan) were evaluated in vitro and in vivo.