The ORF of PaohrR was amplified and subcloned into pET-28a vector between BamHI and XhoI sites. The recombinant protein carried an N-terminal His tag for purification. The cloning was done as reported [13 (link)], and site-directed mutagenesis of the cloned PaohrR was done as reported [13 (link)].
E. coli BL21(DE3) carrying the expression plasmids were cultured in LB medium at 37 °C until OD600nm reached about 0.4–0.6, and then 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added. The temperature was changed to 25 °C for overnight cultivation. Cells were harvested by centrifugation and disrupted through the high-pressure crusher SPCH-18 (Stansted Fluid Power LTD, Harlow, United Kingdom) at 4 °C in ice-cold lysis buffer (50 mM Na2HPO4, 300 mM NaCl and 20 mM imidazole, pH 8.0). The sample was centrifuged and the supernatant was loaded onto the nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Invitrogen, Waltham, MA, USA). The target protein was purified following the manufacturer’s instructions. The eluted protein was loaded onto the PD-10 desalting column (GE) for buffer exchange to 20 mM sodium phosphate buffer (pH 7.6). All protein purification was performed under anaerobic conditions and all buffers used were fully degassed. Purity of the proteins was examined via SDS-PAGE.
E. coli BL21(DE3) carrying the expression plasmids were cultured in LB medium at 37 °C until OD600nm reached about 0.4–0.6, and then 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added. The temperature was changed to 25 °C for overnight cultivation. Cells were harvested by centrifugation and disrupted through the high-pressure crusher SPCH-18 (Stansted Fluid Power LTD, Harlow, United Kingdom) at 4 °C in ice-cold lysis buffer (50 mM Na2HPO4, 300 mM NaCl and 20 mM imidazole, pH 8.0). The sample was centrifuged and the supernatant was loaded onto the nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Invitrogen, Waltham, MA, USA). The target protein was purified following the manufacturer’s instructions. The eluted protein was loaded onto the PD-10 desalting column (GE) for buffer exchange to 20 mM sodium phosphate buffer (pH 7.6). All protein purification was performed under anaerobic conditions and all buffers used were fully degassed. Purity of the proteins was examined via SDS-PAGE.