Glutathione sepharose 4 fast flow affinity column

E. coli BL21 (DE3) harboring the TtCITase-C expression vector was grown in LB medium supplemented with 100 μg/ml ampicillin (Georgiachem, USA) until reaching an optical density of 0.5-0.8 at 600 nm. The E. coli cells were then induced by the addition of 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (Sigma-Aldrich Chemical Co.) at 18°C for 18 h. The induced cells were harvested by centrifugation at 6,120 ×g for 10 min and resuspended in lysis buffer (20 mM sodium phosphate buffer pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM EDTA, or 1 mg/ml lysozyme). The cells were disrupted by sonication (SONICS, 10 s, 40% amplification) at 4°C and the cellular debris was removed by centrifugation at 16,200 ×g for 10 min. After centrifugation, the crude lysate was passed through a Glutathione Sepharose 4 Fast Flow affinity column (GE Healthcare). The column was washed with 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl. Subsequently, the recombinant TtCITase-C was eluted with 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM reduced glutathione. The protein concentration was determined according to the Bradford method with bovine serum albumin as the standard [20] . The final purified recombinant protein was resolved using 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; SMOBIO Technology Inc., Taipei).