Protein expression, purification and enzyme activity analysis
The CDS of BnGSTU12 was cloned into the BamHI and XhoI site of expression vector, pET-28a (+). The constructed vector was transferred into E. coli strain, and recombinant protein was expressed under 0.5 mM IPTG at 28 °C. Recombinant protein fused with 6*His tag was purified by Ni Sepharose 6Fast Flow (Solarbio, Beijing, China) followed with manufacturer’s manual. The protein solution with 6*protein loading buffer was analyzed using 15% SDS-PAGE. The concentration of purified protein was determined according to the BCA Protein Assay Kit (Coolaber, Beijing, China). The GST enzyme activity was performed using GST activity test Kit (Solarbio, Beijing, China). The GST activity was calculated according to the manufacturer’s instructions and repeated three times.