Scorpion insect neurotoxin LqhIT2 is a promising oral biopesticide: high‐level preparation in Pichia pastoris and bioactivity assays

Abstract BACKGROUND Discovering insecticidal proteins with high activity and strict insect specificity and applying them to the biological control of insect pests is of great significance. Oral LqhIT2 has insecticidal activity, which most other insecticidal neurotoxin proteins do not have, but the large‐scale preparation of the toxin is difficult and one of the obstacles to determining its anti‐insect potential for biological control. RESULTS In this study, the expression level of recombinant LqhIT2 (rLqhIT2) in Pichia pastoris was as high as 1.4 g/L under high‐cell‐density fermentation conditions. A purified rLqhIT2 at a final concentration of 0.5 ng/mL inhibited the proliferation of insect Sf9 cells, with typical insect cell oncosis observed, while 200 ng/mL rLqhIT2 was non‐toxic to mammalian cells. The injection of silkworms caused the typical depressive symptoms of flaccid paralysis and death. At a dose of 1 μg/g body weight, 46.7% of the silkworms died within 48 h after injection, and their mortality rate was related to the injection dose. Feeding purified rLqhIT2 to fifth instar Pieris rapae larvae demonstrated its oral insecticidal activity, with a mortality rate 66.7% after 3 days of feeding at a concentration of 10 μg/cm 2 cabbage leaf. CONCLUSION The rLqhIT2 has high insect cells specificity and insecticidal activity when injected and or delivered orally, and may have better application prospects than other reported insect neurotoxins. We describe a method for large‐scale production of functional rLqhIT2 by fed‐batch fermentation using Pichia pastoris , which will further facilitate its application in biological control of agricultural insect pests. © 2024 Society of Chemical Industry.