Cloning and Purification of CalR and ToxR

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.