High-level expression and purification of active scorpion long-chain neurotoxin BjαIT from Pichia pastoris

Abstract

As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pastoris transformants showing high levels of secretory recombinant (r)BjαIT. Secreted rBjαIT was expressed at levels as high as 340 mg/L following methanol induction in a fed-batch reactor, with ∼21 mg of pure rBjαIT obtained from 200-mL fed-batch culture supernatant by Ni2+-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Injection of purified rBjαIT induced neurotoxicity symptoms in locust (Locusta migratoria) larvae, and the half-lethal dose of rBjαIT for locusts at 24-h post-injection ranged from 11 to 14 μg/g body weight. These results demonstrated that large amounts of active rBjαIT were efficiently prepared from P. pastoris, suggesting this system as efficacious for determining rBjαIT insecticidal activity against other agricultural insect pests.