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，如果与受体的结合亲和力提高，则理论上活性应该提高。来自苏云金B.thuringiensis的杀虫毒素（Cry toxins）是AB型毒素，其已被用作有机杀虫剂和用作转基因植物开发的来源。在本报告中，我们描述了一种用于Cry毒素定向进化的方法。该方法可用于增加Cry毒素对其在靶标昆虫害虫中的受体的结合亲和力。使用商业化的T7噬菌体展示系统，我们在噬菌体表面上表达Cry 1Aa毒素作为与衣壳蛋白10 B的融合物。这些重组蛋白结合到一个钙粘蛋白样蛋白，这是一个Cry 1Aa毒素受体的模型目标昆虫家蚕。表达Cry 1Aa的噬菌体对受体的亲和力高于表达Cry 1Ab的噬菌体。使用包被有钙粘蛋白样蛋白的磁珠从T7野生型大肠杆菌中有效地分离表达毒素的大肠杆菌。从表达Cry 1Ab的噬菌体的混合悬浮液中分离表达Cry 1Aa的噬菌体，并浓缩至1.3 × 104<5->倍。最后，在Cry 1Aa毒素结构域2的氨基酸残基369-375处进行随机突变，突变的毒素在噬菌体上表达，并且用钙粘蛋白样蛋白包被的珠对所得噬菌体文库进行5次筛选。结果，排除表达异常或低亲和力突变毒素的菌株，并选择具有高亲和力突变毒素的菌株。其中一种毒素对B.mori的杀虫活性比野生型Cry 1Aa毒素提高了4倍。这些结果表明，将T7噬菌体展示与使用钙粘蛋白样蛋白包被的磁珠的选择相结合的方法可用于增加来自细菌的容易获得的低活性Cry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