Purification of Hexahistidine-tagged E. coli FadR

The hexahistidine-tagged E. coli FadR proteins were produced in E. coli BL21 (DE3) carrying the appropriate expression plasmids (e.g., pET28-fadRec, Table 1) by induction of bacterial cultures at an OD_{600 nm} of 0.8–1.0 with 0.3 mmol/L IPTG at 30°C for 3 h (Feng & Cronan, 2011b (link), Feng & Cronan, 2010 (link)). The cells were pelleted, washed twice with ice-cold PBS buffer (101.4 mmol/L Na₂HPO₄, 1.8 mmol/L KH₂PO₄, 137 mmol/L NaCl, 2.7 mmol/L KCl, 8% glycerol, pH7.4), dissolved in the same buffer and lysed using a French Press. The extracts were centrifuged to remove bacterial debris and the supernatants loaded onto a nickel chelate column (Qiagen). Following washing with ten column volumes of PBS buffer containing 50 mmol/L imidazole, the FadR proteins were eluted with 150 mmol/L imidazole. Appropriate eluted protein fractions were pooled and dialyzed against PBS buffer, then concentrated by ultrafiltration (30 kDa cut-off, Amicon Ultra) (Feng & Cronan, 2010 (link)). The protein purity was judged by 12% SDS-PAGE, followed by staining with Coomassie brilliant blue R250 (Sigma, St. Louis, MO).