Hiprep sephadex g 25 desalting columns

Deca-histidine-tagged NasR was cultured in 2xYT and expression induced in cells at A₂₆₀= 0.5 with 1 mM IPTG at room temperature for 18 hours. The cell pellet was resuspended in resuspension buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1 mM MgCl₂, 2 mM β-ME, 1 mM PMSF, 2 U DNase, and 0.5 mg/mL lysozyme). The cell suspension was then disrupted bead-beating or sonication for intervals of 1 minute on, 1 minute off, on ice, for 5 total minutes of disruption. After cell disruption cell lysates were clarified by centrifugation at 12,000 × G for 30 minutes. The clarified supernatant was passed over Ni-NTA resin columns (Thermo Scientific, GE Healthcare), followed by 6 column-volumes (CV) wash buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1 mM MgCl₂, 35 mM Imidazole). The protein was eluted in 3 fractions of 1 CV elution buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1 mM MgCl₂, 250 mM Imidazole). Eluted protein was dialyzed against dialysis buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 1 mM MgCl₂) for three sequential two-hour buffer exchanges, or alternately buffer exchanged using HiPrep Sephadex G-25 Desalting columns (GE Healthcare). All steps were performed either on ice or at 4°C. The purity of NasR was judged by 4–20% SDS/PAGE followed by Coomassie-staining.