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

苏云金芽孢杆菌产生Cry毒素，在产孢时诱导产生杀虫蛋白。从理论上讲，通过改变毒素的氨基酸序列，特别是受体结合区的氨基酸序列，增加毒素对受体的亲和力，可以使毒素的活性得到改善。在这项研究中，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域，我们首先通过定点诱变构建了13个半胱氨酸取代的突变毒素DNA。在产生突变毒素后，我们利用半胱氨酸的硫醇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，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，展示了在环2、环3或环α 8上氨基酸改变的突变型Cry毒素。采用生物筛选法和磁珠法制备了Cry毒素突变体。在一些文库中，观察了一些显示突变毒素的噬菌体的浓度。虽然我们现在正在确认每种突变毒素的实际杀虫活性和与受体结合亲和力的改善，但我们期望这些突变体具有改善的杀虫活性。

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.