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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 |
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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.
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Received: 09 May 2005
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Corresponding Authors:
WANG Yu-gong
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