Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen
Engineering a Polyspecific Pyrrolysyl-tRNA Synthetase by a High Throughput FACS Screen
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DOI：
10.1038/s41598-019-48357-0
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发表时间：
2019-08-19
影响因子：
4.6
通讯作者：
Eppinger, Jorg
中科院分区：
文献类型：
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作者：
Hohl, Adrian;Karan, Ram;Eppinger, Jorg
pyrlyyl -tRNA合成酶（PylRS）及其同源tRNA（Pyl）被广泛用于向细菌和真核细胞的遗传密码中添加非规范氨基酸（ncAAs）。然而，新的ncaa通常需要一个繁琐的从头工程过程来生成合适的PylRS/tRNA（Pyl）对。我们在此报告了一种预测具有新特性的PylRS变体的策略。设计的多特异性PylRS变体HpRS可催化31种结构不同的ncaa的氨基酰化，这些ncaa具有可点击性、氟化、荧光化和首次生物素化的实体。此外，我们还展示了通过结合生物素的纳米体的位点特异性和无铜偶联策略。多特异性PylRS变体的设计为现有的筛选方法提供了一个有吸引力的替代方案，并为复杂的PylRS-底物相互作用提供了见解。
The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA(Pyl) are extensively used to add noncanonical amino acids (ncAAs) to the genetic code of bacterial and eukaryotic cells. However, new ncAAs often require a cumbersome de novo engineering process to generate an appropriate PylRS/tRNA(Pyl) pair. We here report a strategy to predict a PylRS variant with novel properties. The designed polyspecific PylRS variant HpRS catalyzes the aminoacylation of 31 structurally diverse ncAAs bearing clickable, fluorinated, fluorescent, and for the first time biotinylated entities. Moreover, we demonstrated a site-specific and copper-free conjugation strategy of a nanobody by the incorporation of biotin. The design of polyspecific PylRS variants offers an attractive alternative to existing screening approaches and provides insights into the complex PylRS-substrate interactions.