Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli.
Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli.
复制标题
重编程起始密码子和无义密码子，同时将三种不同的非规范氨基酸安装到大肠杆菌的蛋白质中。
DOI：
10.1007/978-1-0716-3251-2_7
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Tharp,JefferyM
中科院分区：
文献类型：
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作者：
Jiang,Han-Kai;Tharp,JefferyM
多个非规范氨基酸可以被安装到蛋白质中。大肠杆菌使用相互正交的氨酰-tRNA合成酶和tRNA对。在这里，我们描述了一个协议，同时安装三个不同的非典型氨基酸到蛋白质的位点特异性生物共轭在三个网站。该方法依赖于工程化的UAU抑制起始tRNA，其通过詹氏甲烷醛球菌（Methanocaldococcus jannaschiityrosyl-tRNA synthetase）用非规范氨基酸氨酰化。利用这个起始tRNA/氨酰-tRNA合成酶对，以及来自Mazei甲烷八叠球菌和Ca.Methanomethylophilus alvus的吡咯赖氨酰-tRNA合成酶/tRNAPyl对，三个非经典氨基酸可以响应于UAU、UAG和UAA密码子被安装到蛋白质中。
Multiple noncanonical amino acids can be installed into proteins inE. coliusing mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites. This method relies on an engineered, UAU-suppressing, initiator tRNA, which is aminoacylated with a noncanonical amino acid byMethanocaldococcus jannaschiityrosyl-tRNA synthetase. Using this initiator tRNA/aminoacyl-tRNA synthetase pair, together with the pyrrolysyl-tRNA synthetase/tRNAPylpairs fromMethanosarcina mazeiand Ca.Methanomethylophilus alvus, three noncanonical amino acids can be installed into proteins in response to the UAU, UAG, and UAA codons.