An Orthogonal Tyrosyl-tRNA Synthetase/tRNA Pair from a Thermophilic Bacterium for an Expanded Eukaryotic Genetic Code
An Orthogonal Tyrosyl-tRNA Synthetase/tRNA Pair from a Thermophilic Bacterium for an Expanded Eukaryotic Genetic Code
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来自嗜热细菌的正交酪氨酰-tRNA 合成酶/tRNA 对用于扩展真核遗传密码
DOI：
10.1021/acs.biochem.9b00757
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Liu Tao
中科院分区：
文献类型：
--
作者：
Qin Xuewen;Tang Hongting;Cao Wenbing;Dai Zhen;Hu Liming;Huang Yujia;Liu Tao
大肠杆菌衍生的酪氨酰-tRNA合成酶是第一个在真核系统中用于遗传密码扩展的酶，但只能充电有限的一组结构简单的非规范氨基酸。相比之下，嗜热Methanocaldococcus jannaschii衍生的酪氨酰-tRNA合成酶突变体，仅用于原核系统中，由于其显著的耐受突变的结构能力，可以使一组令人惊讶的结构多样的ncAA。受此启发，我们从嗜热脂肪土芽孢杆菌（Geobacillusstearothermophilus）中鉴定了一类新的酪氨酰-tRNA合成酶/tRNATyrpairs，它与大肠杆菌（E.但具有较好的热稳定性。这种新的对在哺乳动物细胞和酵母菌中都是正交的，用于遗传密码扩展，并且与其E相比，可以以相当的细胞效率、更好的特异性和更低的背景来充电各种各样的ncAA。大肠同源物这种热稳定的酶提供了一个替代的支架合成酶库筛选或进化，在真核细胞中遗传编码更复杂的结构ncAA。
TheEscherichia coli-derived tyrosyl-tRNA synthetase was the first enzyme engineered for genetic code expansion in a eukaryotic system but can charge only a limited set of structurally simple noncanonical amino acids. In contrast, the thermophilicMethanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase mutants, used in only a prokaryotic system, can charge a surprisingly large set of structurally diverse ncAAs, due to their remarkable structural ability to tolerate mutations. Inspired by this, we characterized a new class of tyrosyl-tRNA synthetase/tRNATyrpairs from thermophilic bacteriumGeobacillus stearothermophilus, which is homologous to theE. colityrosyl-tRNA synthetase but with better thermostability. This new pair is both orthogonal in mammalian cells and inSaccharomyces cerevisiaefor genetic code expansion and can charge a diverse set of ncAAs with a comparable cellular efficiency, better specificity, and lower background, as compared to those of itsE. colihomologue. This thermostable enzyme provides an alternative scaffold for synthetase library screening or evolution to genetically encode more structurally complex ncAAs in eukaryotic cells.