Ni2 sepharose 6 fast flow beads

The pET-21b(+)-NemR^C106 plasmid^{44 (link)}, which contains only one cysteine (Cys106), was transformed in E. coli BL21 (DE3) cells. Cells were grown in LB supplemented with 50 µg/ml of kanamycin at 37 °C until the A₆₀₀ reached 0.8. IPTG (0.5 mM) was used for the expression induction, followed by 3 h of incubation at 37 °C. Harvested cells were then pelleted at 4 °C, 5000 rpm for 15 min using the Avanti^® J-26xp centrifuge (Beckman Coulter^®) with a JLA-8.1000 rotor and resuspended in lysis buffer composed of 50 mM Tris/HCl pH 8, 0.2 M NaCl, 1 mM DTT, 0.1 mg/ml AEBSF, 1 μg/ml Leupeptin, 50 μg/ml Dnase I, and 20 mM MgCl₂. Cells were disrupted and centrifuged as mentioned above. The supernatant was in-batch incubated with Ni²⁺-Sepharose 6 Fast Flow beads (Cytiva) equilibrated with 50 mM Tris/HCl pH 8, 0.2 M NaCl and 1 mM DTT for 1 h at 4 °C. The beads were packed in a column, and the AKTA™ Pure system (GE Healthcare, Life Sciences) controlled by UNICORN 6.3.0.731 software was used for purification. NemR^C106 was eluted using a linear gradient with elution buffer consisting of 50 mM Tris/HCl pH 8, 0.2 M NaCl, 1 mM DTT and 0 to 700 mM (0–100%) imidazole. Following purification, protein purity was assessed on a non-reducing SDS-PAGE gel, and the pure fractions were dialyzed (~20 ml sample/2L dialysis buffer) overnight at 4 °C against the binding buffer and stored at −20 °C.