Talon cellthru co2 chelating resin

Escherichia coli BL21(DE3) cells harboring pET-his-itaRT were grown in LB media to an OD₆₀₀ of 0.8, and induced with 1 mM IPTG for 16 h at 18°C. Cells were harvested by centrifugation and lysed by sonication in 50 ml of lysis buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 0.5 mM PMSF). The lysate clarified by centrifugation was incubated with 1 ml of Talon CellThru Co²⁺-chelating resin (Takara-Clontech) with agitation for 3 h at 4°C. The resin was washed with 100 ml of wash buffer (50 mM Tris–HCl, pH 7.5; 500 mM NaCl, 10 mM imidazole) and the ItaRT complex anchored at the resin by 6xHis-tagged ItaR was treated with 10 ml of denaturing buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 5 M guanidine-HCl). The denatured ItaT dissociated from the ItaRT complex was separated from the resin and refolded by dialysis at 4°C for 16 h in 2 L of dialysis buffer (25 mM Tris–HCl, pH 7.5; 25 mM NaCl, 1 mM DTT, 5% glycerol). Ca. 50% of ItaT remained soluble after dialysis, the rest precipitated and was discarded. The concentration of soluble ItaT was determined by Bradford method. ItaT was flash-frozen and stored at −80°C.

Escherichia coli BL21(DE3) cells carrying pET22-strep-itaRT-his plasmid were grown to OD₆₀₀ of ∼0.6 in 20 ml of LB medium containing ampicillin (50 μg/ml). Protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG), followed by growth at 18°C for 16 h. Cells were harvested by centrifugation, washed with BW buffer (20 mM Tris–HCl, pH 8.0; 300 mM NaCl) and resuspended in 2 ml of BW buffer supplemented with 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 1 mg/ml lysozyme, and 1 mM β-mercaptoethanol. The cells were lysed by sonication, and the debris was removed by centrifugation. The clarified cell lysate was mixed with 100 μl of Talon CellThru Co²⁺-chelating resin (Takara-Clontech, USA) pre-equilibrated with BW buffer and incubated with gentle rotation at 4°C for 2 h. The resin was washed with BW buffer, and the ItaRT complex was eluted with 1 ml of EB buffer (50 mM Tris–HCl, pH 8.0; 0.5 M imidazole, 300 mM NaCl). The eluted material was combined with 100 μl of Strep-Tactin Superflow Plus resin (Qiagen, USA) and incubated with gentle rotation at 4°C for 4 h. The resin was washed with BW buffer and eluted with 1 ml of BW buffer containing 2.5 mM desthiobiotin (Sigma-Aldrich, USA). Protein fractions were analyzed using Laemmli 12% SDS-PAGE. The gel was stained with EZblue protein stain (Sigma-Aldrich, USA) according to the manufacturer's protocol.