Monoq hr10 10

E. coli BL21 (DE3) cells containing pET30-sigE (gift from Dr Rodrigue) were grown in 1L LB (containing 50 μg/ml Kanamycin) at 37°C till OD₆₀₀ reached 0.5. Protein production was induced by adding 0.5 mM IPTG, followed by growing them for 3 h at 37°C. Cells were harvested by centrifugation (6000 rpm, 10 min, 4°C), resuspended in 20 ml buffer (50 mM Tris-Cl, 200 mM KCl, 10 μM ZnCl₂, 5 mM βME, 1 mM PMSF, 20% Glycerol) and disrupted by sonication. The lysates were spun at 25,000 rpm for 30 min at 4°C. The supernatant was loaded onto Ni-NTA column pre-equilibrated with the above buffer and protein was eluted at 400 mM imidazole. The protein was further purified on MonoQ HR10/10 in Akta purifier (GE Healthcare) using a 0.2–1.0 M NaCl gradient in buffer (20 mM Tris-Cl and 5% Glycerol). The purified protein was concentrated and kept at −80°C after adding equal volume of glycerol.