用于非天然氨基酸遗传编码的大肠杆菌中正交吡咯赖氨酸-tRNA合成酶的定向进化
Directed Evolution of Orthogonal Pyrrolysyl-tRNA Synthetases in Escherichia coli for the Genetic Encoding of Noncanonical Amino Acids.
作者信息
Schmidt Moritz J, Summerer Daniel
机构信息
University of Konstanz, Constance, Germany.
Department of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany.
出版信息
Methods Mol Biol. 2018;1728:97-111. doi: 10.1007/978-1-4939-7574-7_5.
The directed evolution of orthogonal aminoacyl-tRNA synthetases (aaRS) for the genetic encoding of noncanonical amino acids (ncAA) has paved the way for the site-specific incorporation of >170 functionally diverse ncAAs into proteins in a large number of organisms [1, 2]. Here, we describe the directed evolution of orthogonal pyrrolysyl-tRNA synthetase (PylRS) mutants with new amino acid selectivities from libraries using a two-step selection protocol based on chloramphenicol and barnase reporter systems. Although this protocol focuses on the evolution of PylRS variants, this procedure can be universally employed to evolve orthogonal aaRS.
用于非天然氨基酸（ncAA）遗传编码的正交氨酰-tRNA合成酶（aaRS）的定向进化，为在大量生物体中将170多种功能多样的非天然氨基酸位点特异性掺入蛋白质铺平了道路[1,2]。在此，我们描述了使用基于氯霉素和芽孢杆菌RNA酶报告系统的两步筛选方案，从文库中定向进化出具有新氨基酸选择性的正交吡咯赖氨酰-tRNA合成酶（PylRS）突变体。尽管该方案侧重于PylRS变体的进化，但该程序可普遍用于进化正交aaRS。
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