Purification of Hexahistidine-tagged CrlTA Protein Complexes

Protein CrlTA complexes with a hexahistidine tag at the N-terminus of the CrlTA complex without any tag were purified using E. coli BL21 with pET28b-His-crlTA and pET28b-crlTA. Strains were grown in LB with kanamycin (50 μg/ml) and were induced with 1 mM IPTG at OD₆₀₀ ~ 0.1 for 5 h. Cells were collected and resuspended in lysis buffer [50 mM potassium phosphate buffer (pH 8.0), 300 mM NaCl, and protease inhibitor cocktail (Sigma-Aldrich, United States)]. Samples were sonicated using a Sonic Dismembrator (Ningbo Dongzhi, China) at level 2 for 5 min on ice. Ni-NTA resin (Qiagen) was used according to the manufacturer’s protocol. Purified proteins were desalted by passage on disposable Sephadex G-25 prepacked PD-10 columns pre-equilibrated in 20 mM Tris–HCl buffer (pH 8.0), and the protein concentration was measured by the Bi Yuntian BCA assay kit (Haimen, Jiangsu, China). Tricine–SDS-PAGE was performed as previously described (Baba et al., 2006 (link)). A total of 20 μg of protein from each sample was loaded for Tricine–SDS-PAGE.