Cloning and Purification of CsGSTo Proteins

The nucleotide sequences of CsGSTo1 (ANK78262) and 2 (ANK78263) were amplified from an adult C. sinensis cDNA library. The sequences were verified by sequencing, cloned into pET-28a(+) vector (Novagen, Madison, WI, USA) and transformed into Escherichia coli BL21 (DE3) (Thermo Fisher Scientific, Waltham, MA, USA). Bacterial cells were cultured with Luria–Bertani medium supplemented with kanamycin (50 µg/mL). Recombinant proteins, induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside, were purified by a nickel–nitrilotriacetic acid column (Qiagen, Hilden, Germany), followed by thrombin cleavage. The proteins were delipidated using an Octyl-Sepharose 4 Fast Flow column (GE Healthcare, Little Chalfont, UK) and bacterial endotoxin was removed using the Endotoxin Removal System (GenScript, Piscataway, NJ, USA). The homogeneity of the recombinant proteins was monitored by 15% reducing SDS-PAGE. Enzyme activity was assayed using dehydroascorbate substrate [13 (link)].