Recombinant production of the insecticidal scorpion toxin BjαIT in Escherichia coli
Abstract Scorpion long-chain insect neurotoxins have important potential application value in agricultural pest control. The difficulty of obtaining natural toxins is the major obstacle preventing analyses of their insecticidal activity against more agricultural insect pests. Here we cloned the insect neurotoxin BjαIT gene into the pET32 expression vector and expressed the resulting thioredoxin (Trx)-BjαIT fusion protein in Escherichia coli . Soluble Trx-BjαIT was expressed at a high level when induced at 18 °C with 0.1 mM isopropyl β- D -1-thiogalactopyranoside, and it was purified by Ni 2+ -nitriloacetic acid affinity chromatography. After cleaving the Trx tag with recombinant enterokinase, the digestion products were purified by CM Sepharose FF ion-exchange chromatography, and 1.5 mg of purified recombinant BjαIT (rBjαIT) was obtained from 100 ml of induced bacterial cells. Injecting rBjαIT induced obvious neurotoxic symptoms and led to death in locust ( Locusta migratoria ) larvae. Dietary toxicity was not observed in locusts. The results demonstrate that active rBjαIT could be obtained efficiently from an E. coli expression system, which is helpful for determining its insecticidal activity against agricultural insect pests. Highlights Production of soluble Trx-rBjαIT in E. coli. Description of Trx fragment cleavage and isolation of rBjαIT. Purified rBjαIT had insecticidal activity by injection.
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