*4.4. Expression and Purification of Mutant Proteins*

Colonies surrounded by white halos on the purple background of the plate were picked and cultured in LB medium (with 25 μg/mL kanamycin) in a shaking incubator at 37 ◦C. Protein expression was induced with 0.5 mM IPTG at an original OD600 of 0.5–0.7 at 16 ◦C for 12 h. Cells were harvested via centrifugation at 12,000 rpm for 10 min at 4 ◦C. Cell pellets were resuspended gently in binding buffer (20 mM Tris-HCl with 10 mM imidazole, 0.5 M NaCl, pH 8) and disrupted by sonication on ice. The cell debris was removed via centrifugation at 12,000 rpm for 10 min at 4 ◦C, and the supernatants were filtered through a 0.22 μm pore-size filter. Before they were loaded onto NTA-Ni (Qiagen) columns, the supernatants were confirmed using a CV026 plate assay previously described by McClean [42]. Then, the mutant proteins were eluted using a specific wash buffer (20 mM Tris-HCl containing different amounts of imidazole, 0.5 M NaCl, pH 8) from the NTA-Ni columns and evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
