近日,浙江大学化学工程与生物工程学院于浩然研究团队与伦敦大学学院Paul A. Dalby教授课题组合作撰写的综述性论文“Hot spots-making directed evolution easier”发表于国际生物技术顶级期刊Biotechnology Advances(IF14.2)。
为了加速蛋白质的进化,定向进化已经成为一种强大的策略,用于设计提升蛋白的特定性质。然而利用易错PCR,DNA shuffling等传统方法构建的突变体文库通常规模非常大,在没有高通量筛选方法的情况下,筛选这种文库是费时、费力和费钱的。另一方面,以结构或序列信息为指导的点饱和突变也已成为蛋白质工程的一种常用方法,该方法旨在构建“小而精”的突变体文库,进一步提高了定向进化的效率。该方法的前提是突变靶点预测是可靠的,突变靶点或热点位置(Hot spots)的预测对于文库的质量和定向进化的效果至关重要。
该项工作概述了可用于蛋白质进化的七种热点位置,主要分为两类:Ⅰ)基于序列的热点,包括CbD (conserved but different) 位点和共进化残基;Ⅱ)基于三维结构的热点,包括活性位点残基、通道位点、灵活位点、与活性中心“耦合”的远端位点和界面位点。热点位置的选择应基于所需改造的蛋白质特性和可用的结构信息进行(表1)。值得注意的是,这些热点只提供了潜在的突变目标,在实际情况中可能需要其他标准来缩小突变位点的范围。该项工作还对预测这些热点位置的计算工具及将其用于酶工程的许多成功案例进行了综述。
论文以浙江大学化学工程与生物工程学院为第一完成单位,论文中相关研究工作得到国家自然科学基金项目(22108245)的资助。
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