Kasman L M, Lukowiak A A, Garczynski S F, McNall R J, Youngman P, Adang M J
Department of Entomology, University of Georgia, Athens, Georgia 30602, USA.
Appl Environ Microbiol. 1998 Aug;64(8):2995-3003. doi: 10.1128/AEM.64.8.2995-3003.1998.
Activated forms of Bacillus thuringiensis insecticidal toxins have consistently been found to form insoluble and inactive precipitates when they are expressed in Escherichia coli. Genetic engineering of these proteins to improve their effectiveness as biological pesticides would be greatly facilitated by the ability to express them in E. coli, since the molecular biology tools available for Bacillus are limited. To this end, we show that activated B. thuringiensis toxin (Cry1Ac) can be expressed in E. coli as a translational fusion with the minor phage coat protein of filamentous phage. Phage particles displaying this fusion protein were viable, infectious, and as lethal as pure toxin on a molar basis when the phage particles were fed to insects susceptible to native Cry1Ac. Enzyme-linked immunosorbent assay and Western blot analysis showed the fusion protein to be antigenically equivalent to native toxin, and micropanning with anti-Cry1Ac antibody was positive for the toxin-expressing phage. Phage display of B. thuringiensis toxins has many advantages over previous expression systems for these proteins and should make it possible to construct large libraries of toxin variants for screening or biopanning.
苏云金芽孢杆菌杀虫毒素的活化形式在大肠杆菌中表达时，一直被发现会形成不溶性且无活性的沉淀物。由于可用于芽孢杆菌的分子生物学工具有限，若能在大肠杆菌中表达这些蛋白质，将极大地促进对其进行基因工程改造以提高其作为生物杀虫剂的有效性。为此，我们证明活化的苏云金芽孢杆菌毒素（Cry1Ac）可以在大肠杆菌中作为与丝状噬菌体的次要噬菌体外壳蛋白的翻译融合蛋白来表达。当将展示这种融合蛋白的噬菌体颗粒投喂给对天然Cry1Ac敏感的昆虫时，这些噬菌体颗粒是有活力的、具有感染性的，并且在摩尔基础上与纯毒素一样具有致死性。酶联免疫吸附测定和蛋白质印迹分析表明，融合蛋白在抗原性上与天然毒素相当，并且用抗Cry1Ac抗体进行微淘选时，表达毒素的噬菌体呈阳性。苏云金芽孢杆菌毒素的噬菌体展示相对于这些蛋白质以前的表达系统具有许多优势，并且应该能够构建毒素变体的大型文库用于筛选或生物淘选。
