Talon chromatography resin

Cloning and expression of MTb Pck has been previously described [9 (link)]. Pck (Rv0211) with an N-terminal His-tag in pET15b and all cysteine mutants were expressed in E. coli BL21(DE3). The purification process was performed in batches, followed by size exclusion chromatography. Harvested cells were lysed by multiple freeze-thaw cycles and addition of lysozyme. The cell supernatant was loaded onto Talon^® chromatography resin (Clontech) and gently agitated for 1 h on a shaker at 4°C to allow the His-tagged protein to bind the resin. After incubation, the resin was centrifuged at 700 × g for 5 min, and the supernatant was removed. The resin pellet was washed with 10-bed volumes of buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8) and then with 10-bed volumes of buffer containing 10 mM imidazole. The Pck variants were eluted by sequentially increasing the imidazole concentration (100, 200, and 500 mM) in the elution buffer. Pck-containing fractions were filtered to remove traces of resin, concentrated, and purified on a HiLoad 16/60 FPLC (Superdex 75 pg, GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 7.4, or 20 mM Tris-HCl, 150 mM NaCl, pH 7.4.

Cloning, expression, and purification of Mtb Pck has been described [16 (link)]. Briefly, Pck (Rv0211) containing an N-terminal His-tag was expressed in E. coli BL21(DE3). Harvested cells were lysed by multiple freeze-thaw cycles and addition of lysozyme. The cell supernatant was loaded onto Talon chromatography resin (Clontech), and the column was washed with 20 mM Tris-HCl, pH 7.4, containing 500 mM NaCl and 10 mM imidazole. Pck was eluted by sequentially increasing the imidazole concentration (50, 150, 300, 500, and 800 mM) in the elution buffer. Pck-containing fractions were dialyzed against 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 5 mM 2-mercaptoethanol; concentrated; and purified on FPLC HiLoad 16/60 (Superdex 75 pg, GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 8, 500 mM NaCl. Traces of metal ions were removed using Chelex resin (BioRad) in Pck samles prepared for measurements of activation of Pck by different metal ions or for crystallization experiments. 100 ml of Pck solution was gently shaken with 5 g of Chelex resin for 1 hour at 4°C. The Chelex resin was removed by filtration.