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首页> 外文期刊>Applied and Environmental Microbiology >Investigating the Properties of Bacillus thuringiensis Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology
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Investigating the Properties of Bacillus thuringiensis Cry Proteins with Novel Loop Replacements Created Using Combinatorial Molecular Biology

机译：用组合分子生物学研究新型环置换研究苏云金芽孢杆菌Cry蛋白的性质

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Cry proteins are a large family of crystalline toxins produced by Bacillus thuringiensis. Individually, the family members are highly specific, but collectively, they target a diverse range of insects and nematodes. Domain II of the toxins is important for target specificity, and three loops at its apex have been studied extensively. There is considerable interest in determining whether modifications in this region may lead to toxins with novel specificity or potency. In this work, we studied the effect of loop substitution on toxin stability and specificity. For this purpose, sequences derived from antibody complementarity-determining regions (CDR) were used to replace native domain II apical loops to create “Crybodies.” Each apical loop was substituted either individually or in combination with a library of third heavy-chain CDR (CDR-H3) sequences to create seven distinct Crybody types. An analysis of variants from each library indicated that the Cry1Aa framework can tolerate considerable sequence diversity at all loop positions but that some sequence combinations negatively affect structural stability and protease sensitivity. CDR-H3 substitution showed that loop position was an important determinant of insect toxicity: loop 2 was essential for activity, whereas the effects of substitutions at loop 1 and loop 3 were sequence dependent. Unexpectedly, differences in toxicity did not correlate with binding to cadherins—a major class of toxin receptors—since all Crybodies retained binding specificity. Collectively, these results serve to better define the role of the domain II apical loops as determinants of specificity and establish guidelines for their modification.
机译：Cry蛋白是苏云金芽孢杆菌产生的一大类结晶毒素。个体而言，家庭成员具有很高的特异性，但总的来说，它们针对的是各种昆虫和线虫。毒素的结构域II对于靶标特异性很重要，并且已经对其顶点的三个环进行了广泛的研究。确定该区域的修饰是否可能导致具有新的特异性或效力的毒素引起了极大的兴趣。在这项工作中，我们研究了环取代对毒素稳定性和特异性的影响。为此，使用源自抗体互补决定区（CDR）的序列替换天然结构域II顶端环，以创建“ Crybodies”。每个顶部环被单独取代或与第三条重链CDR（CDR-H3）序列库组合取代，以创建七个不同的Crybody类型。对来自每个文库的变体的分析表明，Cry1Aa框架可以在所有环位置上耐受相当大的序列多样性，但某些序列组合会对结构稳定性和蛋白酶敏感性产生负面影响。 CDR-H3取代表明，环位置是昆虫毒性的重要决定因素：环2对于活性至关重要，而环1和环3处的取代作用取决于序列。出乎意料的是，毒性差异与与钙黏着蛋白（一种主要的毒素受体）的结合并不相关，因为所有的Crybobody都保留了结合特异性。总体而言，这些结果有助于更好地定义域II顶环作为特异性的决定因素的作用，并建立对其修饰的指导。

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