Ni2 chelating sepharose fast flow column

The cells were collected by centrifugation at 10,000 × g (4°C, 10 min), washed with phosphate buffer (pH 7.0, 50 mM), and disrupted by sonication at 4°C for 6 min (pulsations of 3 s, amplify 90) using a Vibra-Cell^TM 72405 Sonicator (Sonics, Newtown, CT, USA). The lysates were centrifuged at 20,000 × g (4°C, 30 min) to remove the cell debris. As fused with a 6×histidine tag, the expressed enzyme was allowed to be purified by nickel-affinity chromatography. The supernatant was loaded onto a Ni²⁺-chelating Sepharose Fast Flow column (GE Healthcare, Uppsala, Sweden) equilibrated with a binding buffer (50 mM sodium phosphate buffer, 500 mM NaCl, pH 7.5). The unbound proteins were eluted from the column using a washing buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 50 mM imidazole, pH 7.5), and then the recombinant enzyme was eluted by using a elution buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 7.5). To remove imidazole, the collected fractions were dialyzed against sample buffer (50 mM citrate buffer, pH 5.5). After dialysis, the resulting solution was used as the purified enzyme for further studies.