Protein purification.

His6-tagged Fur protein was expressed in E. coli strain BL21(DE3) as follows: bacterial cells were grown in LB at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.5, and expression of those proteins was induced by addition of IPTG (0.5 M) followed by growth at 30°C for 5 h. Cells were harvested, washed, and suspended in buffer A (20 mM Tris [pH 8.0], 150 mM NaCl, 20 mM imidazole). The cells were then disrupted by sonication, and cell debris was removed by centrifugation at 20,000 × g at 4°C for 30 min. The supernatant was applied to a 1.5-ml nickel-nitrilotriacetic acid (Ni-NTA) agarose column equilibrated in buffer A, washed with 25 column volumes of the same buffer, and eluted using a gradient of buffer A and buffer B (20 mM Tris [pH 8.0], 150 mM NaCl, 250 mM imidazole). The fractions were then collected and analyzed by SDS/PAGE, and selected fractions were dialyzed against buffer C (20 mM Tris [pH 8.0], 150 mM NaCl, 10% glycerol).