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Creation of proteinaceous insecticide using the technology of directed evolution against Bacillusthuringiensis insecticidal toxins

利用苏云金芽孢杆菌杀虫毒素定向进化技术研制蛋白质杀虫剂

基本信息

批准号：
18310053
负责人：
RYOICHI Sato
金额：
$ 11.28万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18310053/
关键词：
biotechnology protein microorganism Bacillus thruingiensis insecticide

项目摘要

Bacillus thuringiensis produces Cry toxins, insecticidal proteins which are induced production at the time of the sporulation. It is theoretically possible to make activity improved toxin by increasing affinity of the toxin to the receptor altering amino-acid sequences especially of the receptor-binding region of the toxin. In this research, we used a phage display system, which was developed in our laboratory, and tried to achieve the directed evolution of Cry toxins to increase the affinity to the receptor. To clarify the receptor binding region, first we constructed 13 cysteine-replaced mutant toxin DNA by site-directed mutagenesis. After production of mutant toxins, we utilized the thiol of the cysteine to be bound with some molecules as inhibitors of receptor-binding, and analyzed these mutants whether inhibition occurred or not. From these experiments we concluded that face made by loop 2, loop 3 and loop alpha 8 is important as a receptor binding site. Next we constructed 11 phage libraries displaying mutant Cry toxin with changed amino acids at loop 2, loop 3 or loop alpha 8. Selection of mutant Cry toxin by bio-panning or magnetic beads system was conducted. From some libraries, concentration of some mutant toxin displaying phages was observed. Although we are now confirming the actual insecticidal activity and improvement of binding affinity to the receptor of each mutant toxin, we are expecting that those mutants have improved insecticidal activities.

苏云金芽孢杆菌产生Cry毒素，即在孢子形成时诱导产生的杀虫蛋白。理论上可以通过增加毒素与受体的亲和力，改变氨基酸序列，特别是毒素的受体结合区的氨基酸序列，来制备活性改善的毒素。在本研究中，我们使用了我们实验室开发的噬菌体展示系统，并试图实现Cry毒素的定向进化，以增加对受体的亲和力。为了明确受体结合区，我们首先通过定点突变构建了13个半胱氨酸取代的突变毒素DNA。突变体毒素产生后，我们利用半胱氨酸的巯基与一些分子结合作为受体结合的抑制剂，并分析这些突变体是否发生抑制。从这些实验中，我们得出结论，由环2、环3和环α 8形成的面作为受体结合位点是重要的。接下来，我们构建了11个噬菌体文库，其展示在环2、环3或环α 8处具有改变的氨基酸的突变体Cry毒素。通过生物淘选或磁珠系统进行突变体Cry毒素的选择。从一些文库中，观察到一些展示突变体毒素的浓度。虽然我们现在正在确认每种突变毒素的实际杀虫活性和对受体的结合亲和力的改善，但我们预期这些突变体具有改善的杀虫活性。