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

在ab型毒素中，它由活性结构域和结合结构域组成，理论上，如果与受体的结合亲和力得到改善，活性应该得到改善。苏云金芽孢杆菌的杀虫毒素（Cry毒素）是ab型毒素，已被用作有机杀虫剂和转基因植物开发的来源。在本报告中，我们描述了一种定向进化Cry毒素的方法。该方法可提高Cry毒素与靶害虫受体的结合亲和力。利用商业化的T7噬菌体展示系统，我们将Cry1Aa毒素与衣壳蛋白10B融合在噬菌体表面表达。这些重组噬菌体与一种钙粘蛋白样蛋白结合，该蛋白是模型靶昆虫家蚕中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。利用包被钙粘蛋白样蛋白的磁珠有效地从T7野生型噬菌体中分离出表达毒素的噬菌体。从表达Cry1Ab的噬菌体混合悬液中分离出表达Cry1Aa的噬菌体，并浓缩至1.3 × 10^<5->倍。最后，在Cry1Aa毒素2结构域369 ~ 375个氨基酸残基上进行随机突变，将突变毒素在噬菌体上表达，并用cadherin样蛋白包被小珠对噬菌体文库进行5次筛选。因此，排除表达异常或低亲和力突变毒素的噬菌体，选择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比野生型Cry1Aa毒素提高了4倍。这些结果表明，将T7噬菌体展示与钙粘蛋白样蛋白包被磁珠选择相结合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.