Protein expression was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside Pirfenidone price (IPTG) for 5 h. Cultured cells were harvested by centrifugation

at 5000 g (4 °C, 15 min), resuspended in 25 mM HEPES (pH 7.0) and disrupted via French Pressure Cell at 10 000 psi. Soluble and insoluble fractions of cells were separated by centrifugation at 10 000 g (4 °C, 20 min) and analyzed by sodium dodecyl sulfate (12% w/v) polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentrations were measured via Bradford microassay (Bio-Rad). For preparation of soluble CyaC, the protein was initially purified using a cation-exchange FPLC system (8-mL Mono S column; GE Healthcare). The column was equilibrated with buffer A [25 mM HEPES (pH 7.0), 1 mM 1,4-dithiothreitol]. Chromatographic separations were achieved with an increased step gradient of buffer B (1 M

NaCl in buffer A) via 20% B (5-column volume), 20–30% B (2.5-column volume) and 30–100% B (2.5-column volume). Elution fractions across the 700 mM CX5461 NaCl peak were pooled and subjected to further purification by hydrophobic interaction chromatography (HIC, 5-mL HiTrap™Phenyl HP column). Separation was achieved via a stepwise decrease of 2 M NaCl concentrations in buffer A. Subsequently, the eluted fraction at 2 M NaCl was loaded onto gel filtration (25-mL Superdex™75 column) equilibrated with buffer A Baricitinib at flow rate of 0.4 mL min−1. Peak fractions containing the 21-kDa protein were pooled and concentrated by ultrafiltration using 50-mL Centriprep column (10-kDa cutoff). For preparation of refolded CyaC, insoluble inclusions were washed with 80 mM K2HPO4 (pH 6.5) containing 0.8 M NaCl and 0.1% Triton X-100, followed by washing twice

with cold distilled water. CyaC inclusions (1–5 mg mL−1) were solubilized in 20 mM Tris-HCl (pH 8.0) containing 8 M urea at 37 °C for 1 h. After centrifugation at 18 000 g for 20 min, the unfolded CyaC protein was initially refolded in Superdex™75 column equilibrated with refolding buffer [20 mM Tris-HCl (pH 8.0), 2 M urea, 150 mM NaCl]. The eluted monomeric CyaC fraction was dialyzed against 300 volumes of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and 1 M urea at 4 °C for 4 h, and finally dialyzed twice against the same buffer without urea. Purified CyaC separated by SDS-PAGE (12% gel) was eluted out from the excised gel by soaking with 0.1 M NH4HCO3 and subsequently digested with trypsin at a substrate : enzyme ratio of 10 : 1. Trypsin-generated fragments were separated on a 0.18 × 100-mm C18 column (Thermo Electron) and analyzed by LC/MS/MS (Finnigan LTQ Linear Ion Trap Mass Spectrometer). Toxin activation in vitro mediated by CyaC was performed by mixing 10 μg of purified CyaC monomer with E.