基于自定义多位点半饱和突变技术实现氨酰tRNA合成酶的定向进化

蛋白质作为生命的物质基础对于生物体的生命活动的维持以及生物体内生化活动的调控至关重要。然而目前学界对蛋白质的功能与结构的关系的认知尚浅，无法较为精准地通过基于蛋白质结构理性设计出具有较高生理活性或新颖功能的蛋白质。蛋白质的定向进化则成功地解决了这一难题，蛋白质的定向进化是直接从基因层面施以突变，蛋白质的相应位点的氨基酸残基就会产生突变从而改变蛋白质的结构，产生新的功能，通过设定合适的筛选条件就能得到具有目的功能蛋白质。目前蛋白质定向进化发展的限制因素主要集中在建立高质量的突变文库或者建立高通量的筛选方法方面。这里我们提出了一种多位点自定义半饱和突变技术，此技术是基于寡核苷酸定点突变技术而提出改进的方法。具体原理是基于氨基酸的结构信息和简并密码子在突变时将单个位点的氨基酸残基的多样性降低，在每轮进化过程可接受的文库大小限制下，可以换取对更多突变位点的覆盖，这样将有望突破蛋白质定向进化的“高原效应”从而得到活性更高的蛋白质。此外由于本实验室进行的构建人工酶的可调控催化中心的课题，需要将具有特殊结构的非天然氨基酸（UAA）定点插入至骨架蛋白的特定位点以实现对酶的催化性能调控。所以我们用上述突变策略对实验室自合成的7种UAA进行了筛选，最终通过定向进化成功得到了识别其中3种UAA的氨酰tRNA合成酶。

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