The constructed vector was transformed into DH10Bac E. coli competent cells, and the baculovirus was generated from the pFastBac clones for insect-cell expression following standard procedures. For protein production, Sf9 insect cells were infected with baculovirus for 66 h at a 1:100 virus:cell ratio (volume ratio of virus to sf9 cells). The infected cells were harvested by centrifuge at 200 g for 10 min and the cell pellets were stored in liquid nitrogen.
The cell pellets were resuspended and sonicated in buffer A (20 mM Tris-HCl buffer pH8.0 containing 5 mM MgCl 2 , 300 mM NaCl, 5% glycerol, 0.05% CHAPS and 1 mM DTT) and centrifuged at 40,000 g for 30 min. The recombinant protein was purified from the supernatant with Glutathione Sepharose 4B affinity-chromatography column (GE Healthcare), dialyzed against buffer A, and was further purified with HiTrap Q HP column equilibrated by buffer A, and concentrated to 2 mg/mL in 20 mM Tris-HCl pH 7.5 containing 5 mM MgCl 2 , 150 mM NaCl, 1 mM DTT and 10% glycerol.
The cell pellets were resuspended and sonicated in buffer A (20 mM Tris-HCl buffer pH8.0 containing 5 mM MgCl 2 , 300 mM NaCl, 5% glycerol, 0.05% CHAPS and 1 mM DTT) and centrifuged at 40,000 g for 30 min. The recombinant protein was purified from the supernatant with Glutathione Sepharose 4B affinity-chromatography column (GE Healthcare), dialyzed against buffer A, and was further purified with HiTrap Q HP column equilibrated by buffer A, and concentrated to 2 mg/mL in 20 mM Tris-HCl pH 7.5 containing 5 mM MgCl 2 , 150 mM NaCl, 1 mM DTT and 10% glycerol.