Ni nta column

The Per a 5 gene was subcloned into pFastBac1 vector (Novagen, Madison, WI, USA) using EcoR I and Sal I sites and the resulted construct was transformed into E. coli strain DH10Bac to generate recombinant bacmid. The positive colonies were selected and followed by PCR identification. The recombinant bacmid was transfected into Sf-9 cells by using Cellfectin (Invitrogen Corporation, Carlsbad, USA) and incubated in SF-900II liquid medium (Invitrogen Corporation, Carlsbad, USA) for 5 days at 27°C until the cells got swollen. The supernatant was collected as P1 viral stock. P2 viruses were amplified for later infection. A total of 500 mL of Sf-9 cells were infected by P2 viruses and harvested at 72 h. The cells were lysed against 50 mM Tris-HCl with 300 mM NaCl and 5% glycerol. The supernatant was loaded on Ni-NTA column (Genscript, Nanjing, China), washed with running buffer containing 50 mM Tris-HCl, 300 mM NaCl, and 5% glycerol (pH 8.0) and eluted with elution buffer containing 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, and 5% glycerol (pH 8.0). The eluted fractions were obtained and identified as Per a 5 (iPer a 5). The purified iPer a 5 was dialyzed in carbonate-bicarbonate buffer (0.05 M, pH 9.6) for further investigation. The concentration of iPer a 5 was determined by using a Coomassie Plus assay kit with BSA as standard (Thermo Scientific Pierce, Rockford, IL, USA).