High expression and rapid purification of recombinant scorpion anti-insect neurotoxin AaIT |
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Authors: | Hongbo Li Yuxian Xia |
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Institution: | (1) Genetic Engineering Research Center, College of Bioengineering, Chongqing University, 400030 Chongqing, People’s Republic of China;(2) Chongqing Engineering and Technology Center of Fungal Insecticide, Lab of Functional Gene and Regulation Technologies under Chongqing Municipal Education Commission, 400030 Chongqing, People’s Republic of China |
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Abstract: | Scorpion long-chain insect neurotoxins are potentially valuable as agricultural pest control agents. Unfortunately, natural
insect neurotoxins are limited in quantity and difficult to obtain from scorpion venom. To determine if recombinant insect
neurotoxin is active to insects, we expressed and purified an AaIT fusion protein in Escherichia coli and a recombinant AaIT protein in Pichia pastoris. To quantify AaIT expression in P. pichia colonies, we produced highly sensitive antiserum against AaIT in BALB/c mice. P. pastoris transformants that highly expressed AaIT were selected based on immunoassay with the AaIT antiserum. The P. pastoris recombinant AaIT was rapidly purified in a new and efficient two-step method that eliminated all contaminant proteins using
ultracentrifugal filters with molecular weight cut-off 10 kDa and 3 kDa. With this new protocol 10 mg of purified recombinant
AaIT was harvested from a 1-l P. pastoris culture. Bioactivity tests indicated that the P. pastoris recombinant AaIT was highly toxic to cockroach larvae, but the E. coli AaIT fusion protein was not toxic to cockroaches. The new expression, screening, and purification protocol described here
was efficient for quickly producing high concentrations of pure, bioactive protein. |
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Keywords: | AaIT Expression Purification Antiserum Neurotoxin |
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