Basic research to design "Order-made" Cry toxin

通过突变分析了Cry 4Aa结构域II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化。这表明，多个环协同工作的受体结合可能是分散在结构域II，也许在结构域III的Cry 4Aa。该模型是新的，并且不同于充分表征的Cry 1A毒素。基于这一假设，构建了含有结构域II环2区随机氨基酸序列的突变体Cry 4Aa文库。在通过使用蚊幼虫（尖音库蚊）的生物测定筛选后，分离出了几种可以以新的作用模式杀死蚊子的独特突变体。对这些突变体的研究仍在进行中。在开发有效的害虫治理的研究中，我们发现来自Xestia c-nigrum颗粒体病毒的病毒变蛋白酶Enhancin 3和昆虫特异性蝎毒素AaIT可以用作生物农药来增强Cry毒素的毒性。此外，我们开发了新的肽标签，4AaCter从C-末端区域的Cry 4Aa。我们证明，使用4AaCter可以为重组蛋白的有效生产提供创新策略。

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.