Hitrap q anion exchange chromatography

Cp149 (amino acids 1–149) is a truncated form of the HBV core protein. It was expressed in Escherichia coli and the expressed protein was purified^{56 (link)}. The gene encoding Cp149-Y132A with a C-terminal thrombin cleavage site was synthesized and optimized for expression in bacterial cells (Gene Universal). The gene was cloned between the NdeI and BamHI sites in pET23a vector containing a hexahistidine tag. Recombinant Cp149-Y132A was expressed in E. coli BL21 (DE3) cells that were cultured in LB medium at 37 °C. When the culture reached OD600 of 0.7–0.8, expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside, and the culture was cooled to 16 °C. The cells were harvested after 16 h, flash-frozen in liquid nitrogen, and stored at −80 °C. To purify Cp149-Y132A, thawed cells were resuspended in lysis buffer (20 mM Tris-HCl pH 9.0, 200 mM NaCl, 10 mM imidazole, 10 ug per mL DNaseI, 1 mM phenylmethylsulfonyl fluoride) and sonicated. The supernatant was loaded onto Ni-NTA affinity resin and the protein was eluted with a gradient of 20 to 500 mM imidazole in lysis buffer. After removing the histidine tag with thrombin, the protein was further purified by HiTrap Q anion exchange chromatography (17115401, GE Healthcare).