Basic research to design "Order-made" Cry toxin

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The biological functions of loops in domain II of Cry4Aa were analyzed by mutagenesis. The results showed that all loops can be engineered without a loss of toxicity. This suggested that the multiple loops working cooperatively for receptor binding may be spread out in domain II and perhaps in domain III of Cry4Aa. This model was new, and was different from that of well characterized Cry1A toxins. Based on this hypothesis, mutant Cry4Aa library that contained random amino-acid sequences in domain II loop 2 region was constructed. Upon screening by bioassay using mosquito larvae(Culex pipiens), several unique mutants that may kill mosquitos with novel mode of action were isolated. Investigation to characterize these Cry4Aa mutants is still going on. On the researches to develop efficient insect-pest management, we showed that the virus metaroprotease, Enhancin3 from Xestia c-nigrum granulovirus and the insect-specific scorpion toxin, AaIT can be used as bio-pesticide to enhance the toxicity of Cry toxin. In addition, we developed novel peptide-tag, 4AaCter from the C-terminal region of Cry4Aa. We demonstrated that the use of 4AaCter may provide an innovational strategy for the efficient production of recombinant proteins.