E coli m15 prep4

For 6×His-OxyR^C199S purification, plasmid pIZ1885 was transformed into E. coli M15 [pREP4] (Qiagen, Valencia, CA, USA). M15/pIZ1885 was grown in LB broth containing ampicillin, and expression of 6×His-OxyR^C199S was induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). After 3 h of induction, cells were centrifuged and resuspended in 10 ml of lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM imidazole) per g of pelleted cells, and were lysed by sonication. The suspension was centrifuged at 10 000 rpm for 30 min and the supernatant containing the soluble fraction of 6×His-OxyR^C199S was transferred to a HisTrap HP nickel affinity chromatography column (GE Healthcare, Wauwatosa, WI, USA). The column was washed with 4 ml of lysis buffer, 4 ml of washing buffer (20 mM Tris, 300 mM NaCl, 30 mM imidazole) and 4 ml of the same buffer with 50 mM imidazole. Protein elution was performed with 3 ml of elution buffer (20 mM Tris, 300 mM NaCl, 300 mM imidazole). Elution fractions enriched in 6×His-OxyR^C199S were selected and combined. Imidazole was removed by transferring to an Amicon^® ultra centrifugal filter (Merck Millipore, Darmstadt, Germany) and washing with storage buffer (20 mM Tris, 300 mM NaCl, 10% glycerol) or by dialyzing in cellulose membranes (Sigma-Aldrich, St Louis, MO, USA). 6×His-OxyR^C199S was either used immediately or frozen in liquid nitrogen and stored at −80ºC.