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

苏云金芽孢杆菌杀虫毒素的定向进化，用于制造蛋白质杀虫剂。

• 批准号: 15380228
• 负责人: SATO Ryoichi
• 金额: $ 8.96万
• 依托单位: National University Corporation 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: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15380228/
• 关键词: Bacillus thuringiensis; Cry toxin; phase display; directed evolution; cadherin-like protein; Bacillus thuringiensis; insecticidal protein; phage display; bio-panning

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.

在由活性和结合结构域组成的AB型毒素中，如果改善与受体的结合亲和力，应在理论上改善活性。硫属菌的杀虫剂毒素（哭泣的毒素）是AB型毒素，已被用作有机杀虫剂，也是转基因植物发育的来源。在本报告中，我们描述了一种指导哭泣毒素进化的方法。该方法可用于增加cry毒素对目标虫害中其受体的结合亲和力。使用商业T7噬菌体播放系统，我们在噬菌体表面上表达Cry1aa毒素作为与衣壳蛋白10b的融合。这些重组噬菌体与钙粘蛋白样蛋白结合在一起，该蛋白是模型靶向昆虫bombyx mori中的Cry1aa毒素受体之一。表达CRY1AA对受体的亲和力高于表达Cry1ab的噬菌体。使用涂有钙粘蛋白样蛋白的磁珠从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的噬菌体的混合悬浮液中分离出表达Cry1aa的噬菌体，并以多达1.3 x 10^<5->倍数浓缩。最后，在Cry1aa毒素的结构域2中，在氨基酸残基369-375中进行随机突变，突变毒素在噬菌体上表达，并用钙粘蛋白样蛋白质涂层的珠子筛选了五次所得的噬菌体库。结果，排除表达异常或低亲和力突变毒素的噬菌体，并选择了具有高亲和力突变体毒素的噬菌体。这些毒素对b.mori的一种杀虫活性比野生型Cry1aa毒素增加了四倍。这些结果表明，使用钙粘蛋白样蛋白涂层的磁珠将T7噬菌体显示与选择的方法可用于增加细菌易于获得的低活性哭泣毒素的活性。

项目成果

目的昆虫に効くδ内毒素変異体を選抜するためのシステム

用于选择对目标昆虫有效的δ内毒素突变体的系统

• 发表时间: 2005

Location of the Bombyx mori aminopeptidase N type l binding site on a Bacillus thuringiensis CrylAa toxin.

苏云金芽孢杆菌 CrylAa 毒素上的家蚕氨肽酶 N I 型结合位点的位置。

• 发表时间: 2005
• 期刊: Applied and Environmental Microbiology 71
• 作者: S.Atsumi他 8名

Location of the Bombyx mori Aminopeptidase N type 1 binding site on a Bacillus thuringiensis Cry1Aa toxin.

苏云金芽孢杆菌 Cry1Aa 毒素上家蚕氨基肽酶 N 1 型结合位点的位置。

• 发表时间: 2005
• 期刊: Appl Environ Microbiol. 71(7)
• 作者: Shogo Atsumi;Eri Mizuno;Hirotaka Hara;Kazuko Nakanishi;Madoka Kitami;Nami Miura;Hiroko Tabunoki;Ayako Watanabe;Ryoichi Sato
• 通讯作者: Ryoichi Sato