降低这个句子的重复率：All the genes and corresponding restriction enzyme NcoIXhoI sequences were entrusted to be synthesized by Shanghai Sangon Biotech Shanghai China The synthesized genes were digested with the

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.