Hitrap chelating sepharose columns

The entire coding region of calR was amplified, purified, and cloned between BamHI and HindIII sites of plasmid pET28a (Novagen). The recombinant plasmid encoding His‐CalR was then transformed into E. coli BL21λDE3 cells. Expression of His‐CalR was induced by adding 1 mmol/L IPTG (isopropyl‐b‐D‐thiogalactoside) and purified under native conditions with nickel‐loaded HiTrap Chelating Sepharose columns (Amersham) (Sun et al., 2012). Briefly, cells were collected by centrifugation, and then resuspended in 10 ml of 57 mmol/L sodium phosphate buffer, 500 mmol/L NaCl, 5 mmol/L imidazole, pH 8.0. After being disrupted by a cell cracker at >1000 psi, the insoluble material was pelleted by centrifugation at 10,000 rpm for 30 min under 4°C. The clarified supernatant was loaded onto a 5 ml nickel−chelating column, then washed with five column volumes of wash buffer, and eluted with an imidazole gradient. The eluant containing His‐CalR protein was dialyzed against 0.02 mol/L PBS (41 mmol/L Na₂HPO₄, 5 mmol/L NaH₂PO₄, 145 mmol/L NaCl, and 20% glycerol, pH 8.0), and concentrated to a final concentration of about 0.3−0.6 mg/ml. The purified protein was stored at −80°C, and the protein purity was confirmed by SDS‐PAGE.

The entire coding region of calR or the truncated toxR (1-528 bp, a.a.1-176) was amplified, purified, and cloned into pET28a (Novagen). The recombinant plasmid encoding His-CalR or His-ToxR was then transformed into E. coli BL21λDE3 cells. Expression and purification of His-CalR were similar to that of His-OpaR[41 (link)], while His-ToxR was the same as that of His-AphA [42 (link)]. The purified proteins were concentrated with nickel loaded HiTrap Chelating Sepharose columns (Amersham) and concentrated to a final concentration of about 0.3-0.6 mg/ml. The purified proteins were stored at -80°C, and the protein purity was confirmed by SDS-PAGE.