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.