Purification and Characterization of CtpA Variants

DNA encoding amino acid 38 to the C terminus of CtpA was subcloned into plasmid pET15b between the NdeI and XhoI sites to encode N-terminal His₆-tagged CtpA(ΔN37). Similar plasmids encoding CtpA-S302A, CtpA(ΔC6), CtpA-L426K L430K, or CtpA-L426A L430A were generated by site-directed mutagenesis. For all CtpA proteins, E. coli BL21(DE3) transformants were grown at 37°C to optical density at 600 nm (OD₆₀₀) = 0.6 to 0.7 before being induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 16°C overnight. Cells were lysed by passing through a microfluidizer cell disruptor in 10 mM potassium phosphate, pH 8.0, 10 mM imidazole, 0.25 M NaCl. The homogenate was clarified by centrifuging at 27,000 × g, and the supernatant was applied to a HiTrap-Ni column (GE Healthcare) preequilibrated with lysis buffer. Proteins were eluted with a 10 to 300 mM imidazole gradient in 10 mM potassium phosphate, pH 8.0, containing 0.25 M NaCl. Fractions containing His₆-CtpA were collected. The N-terminal His tag was removed using thrombin (0.5 units/mg) by dialyzing against 20 mM Tris, pH 8.0, 150 mM NaCl overnight at 4°C. Untagged CtpA was further purified with HiTrap-Q in 10 mM Tris, pH 8.0, and a 50 to 500 mM NaCl gradient and polished by gel filtration in 10 mM Tris, pH 8.0, and 150 mM NaCl using Superdex 200 prep-grade column (16 × 600 mm, GE Healthcare).