Basic research to design "Order-made" Cry toxin

通过诱变分析了Cry4Aaⅱ结构域环的生物学功能。结果表明，所有的环都可以在不损失毒性的情况下进行工程设计。这表明协同受体结合的多个环可能分布在Cry4Aa的结构域II和可能分布在结构域III。该模型是一种新模型，不同于已有的Cry1A毒素模型。基于这一假设，构建了包含ⅱ域环2区随机氨基酸序列的突变体Cry4Aa文库。通过蚊幼虫（库蚊）的生物测定筛选，分离出了几种独特的突变体，这些突变体可能以新的作用方式杀死蚊子。对这些Cry4Aa突变体特征的研究仍在进行中。在开发有效的害虫防治研究中，我们发现了毒超蛋白酶、毒毒增强酶3和毒蝎毒素ait可以作为生物农药来增强毒蝎毒素的毒性。此外，我们还从Cry4Aa的c端区开发了新的肽标签4AaCter。我们证明了使用4AaCter可能为高效生产重组蛋白提供了一种创新策略。

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.