Improving the secretory expression of active recombinant AaIT in Pichia pastoris by changing the expression strain...

Scorpion long-chain insect selective neurotoxin AaIT has the potential to be used against agricultural insect pests. However, there is still a lack of a heterologous gene expression system that can express AaIT efficiently. Here, using X33 as the host strain and pPICZαA as the expression vector, one transformant had the highest expression of recombinant AaIT (rAaIT) was obtained, and secreted as high as 240 mg/l rAaIT in fed-batch fermentation. Secretory rAaIT was purified by Ni²⁺-nitriloacetic affinity and CM chromatography, and 8 mg of high purity rAaIT were purified from 200 ml fed-batch fermentation cultures. Injecting silkworm (Bombyx mori Linnaeus) and Galleria mellonella larvae with rAaIT resulted in obvious neurotoxin symptoms and led to death. These results demonstrate that a large amount of anti-insect active rAaIT could be prepared efficiently.