Expression and Bioactivity Assay of Fusion Protein of Cowpea Trypsin Inhibitor(CpTI) and Thioredoxin
CHANG Wen-jun;WANG Yu-gong;LEI Lu-wang
tate Key Laboratory of Tropical Crop Biotechnology,Institute of Tropical Biotechnology,Chinese Academy of Tropical Agricltural Sciences,Haikou 571101,China
Abstract：To construct fusion protein expression vector, cowpea trypsin inhibitor (CpTI) gene was linked with thioredoxin gene in the pTrxFus through a flexible linker designed with the software Chou-Fasman. The recombinant vector pTrxFus-CpTI was identified and transformed into Escherichia coli GI724 to overexpress the fusion protein. The expression was induced with tryptophan. The fusion protein was confirmed to be soluble by SDS-PAGE analysis of the supernatant and precipitate of lysates. SDS-PAGE also showed that expressed protein accounted for about 20% of the total bacterium protein. With Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride(BApNA) as substrate, the bioactivity and heat resistance of fusion protein were assayed and the result showed, like soybean trypsin inhibitor, fusion protein performed relatively high thermal resistance. SDS-PAGE showed most fusion protein remained soluble after treated at 80 ℃, which greatly facilitates its purification and application. Thioredoxin-CpTI could be digested into thioredoxin and CpTI by incubating at 37 ℃ for 16 h with enterokinase. The CpTI inhibition activity was about 80% of thioredoxin-CpTI, indicating that the CpTI stability was increased via the fusion with thioredoxin. This work has laid a foundation for application of thioredoxin-CpTI as a biological pesticide.
