Protein Expression and Purification Protocol using E. coli BL21(DE3)

The genes and corresponding NcoI/XhoI restriction enzyme sequences were synthesized by Shanghai Sangon Biotech (Shanghai, China). After synthesis, the genes were digested with NcoI/XhoI and attached to the pET28a(+) expressed plasmid, with a (His)6-tag introduced at the C-terminus. These plasmids were then transformed into E. coli BL21(DE3) and cultured in Luria-Bertani media supplemented with kanamycin (30 ng/μL) at 37 °C for 12 h. Protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (0.5 mM) and incubated at 17 °C for 16 h. The (His)6-tag recombinant proteins were purified using immobilized metal-ion affinity chromatography, following our previously reported methods. Protein expression and purity were analyzed using SDS-PAGE with a 12% polyacrylamide gel.