Directed evolution of the Bacillus thuringiensis insecticidal toxin for the creation of proteinaceous insecticide.

在AB型毒素中，由活性结构域和结合域组成，理论上，如果与受体的结合亲和力得到改善，活性应该会得到改善。苏云金杆菌产生的杀虫毒素(Cry毒素)是一种AB型毒素，已被用作有机杀虫剂和转基因植物发育的来源。在本报告中，我们描述了一种定向进化Cry毒素的方法。这种方法可用于提高Cry毒素与其受体在靶标害虫中的结合亲和力。利用商品化的T7噬菌体展示系统，我们在噬菌体表面表达了Cry1Aa毒素，并与衣壳蛋白10B融合。这些重组噬菌体与一种类钙粘蛋白结合，该蛋白是模型中的目标昆虫家蚕的Cry1Aa毒素受体之一。表达Cry1Aa的噬菌体对受体的亲和力高于表达Cry1Ab的噬菌体。用包被类钙粘素蛋白的磁珠从T7野生型噬菌体中有效地分离出表达毒素的噬菌体。从表达Cry1Ab的噬菌体混合悬浮液中分离到表达Cry1Aa的噬菌体，浓缩倍数可达1.3×10~(-5)。最后，对Cry1Aa毒素结构域2的369-375位氨基酸进行随机突变，将突变的毒素表达在噬菌体上，并用类钙粘素蛋白包被的小球对噬菌体文库进行5次筛选。结果排除了表达异常或低亲和力突变毒素的噬菌体，筛选出具有高亲和力突变毒素的噬菌体。其中一种毒素对家蚕的杀虫活性是野生型Cry1Aa毒素的4倍。这些结果表明，结合T7噬菌体展示和类钙粘蛋白磁珠选择的方法可以提高细菌中容易获得的低活性Cry毒素的活性。

In AB-type toxins, which consist of active and binding domains, activity should theoretically be improved if binding affinity to the receptor is improved. The insecticidal toxins (Cry toxins) from B.thuringiensis are AB-type toxins that have been used as organic insecticides and as sources for the development of transgenic plants. In the present report, we describe a method for directed evolution of Cry toxin. This method can be used to increase the binding affinity of Cry toxins for their receptors in target insect pests. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Toxin-expressing phages were effectively isolated from T7 wild-type phages using magnetic beads coated with cadherin-like protein. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 1.3 x 10^<5->fold. Finally, random mutations were made in amino acid residues 369-375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened five times with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. The insecticidal activity of one of these toxins against B.mori was increased four-fold over that of wild-type Cry1Aa toxin. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.