Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.
Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.
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DOI：
10.1016/j.jmb.2020.06.014
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发表时间：
2020-07-24
影响因子：
5.6
通讯作者：
Cooley RB
中科院分区：
文献类型：
--
作者：
Beyer JN;Hosseinzadeh P;Gottfried-Lee I;Van Fossen EM;Zhu P;Bednar RM;Karplus PA;Mehl RA;Cooley RB
遗传密码扩增（GCE）技术将非规范氨基酸（ncAA）在琥珀终止密码子处并入蛋白质中。为了避免不需要的截短蛋白和提高ncAA蛋白产量，基因组重新编码的E.缺乏释放因子1（RF 1）的大肠杆菌正成为GCE应用中越来越受欢迎的表达宿主。然而，在不存在RF 1的情况下，内源性近同源琥珀抑制tRNA可导致用天然氨基酸代替ncAA的污染蛋白质形式。在这里，我们表明，第二代氨酰tRNA合成酶（阿尔斯）/tRNACUA对3-硝基酪氨酸的位点特异性掺入不能胜过近同源抑制在RF 1缺陷的表达宿主，因此不能产生均匀硝化蛋白。为了解决这个问题，我们使用Rosetta来靶向硝基Tyr阿尔斯活性位点中的位置以改善底物结合，然后构建小的变体文库以进行标准选择方案。最佳选择的变体具有约2倍的效率，并且值得注意的是，这种相对较小的改进使得能够在RF 1缺陷型表达宿主中均匀掺入硝基Tyr，从而消除了与典型的含RF 1表达宿主相关的截短问题。结构和生化数据表明阿尔斯效率的提高是基于更高的亲和力底物结合。总之，阿尔斯效率的适度改善提供了很大的实际影响，并扩大了我们通过生产同质的、无截断的含硝基酪氨酸蛋白质来研究蛋白质硝化在疾病发展中所起作用的能力。这项工作建立了Rosetta引导的设计和增量阿尔斯改进作为一个可行的和可访问的路径，以改善GCE系统的截断和/或近同源抑制问题的挑战。
Genetic Code Expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of E. coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a 2nd-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.
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影响因子：
3.5
作者：
Heinemann IU;Rovner AJ;Aerni HR;Rogulina S;Cheng L;Olds W;Fischer JT;Söll D;Isaacs FJ;Rinehart J
通讯作者：
Rinehart J
影响因子：
5.5
作者：
Nuriel T;Whitehouse J;Ma Y;Mercer EJ;Brown N;Gross SS
通讯作者：
Gross SS
DOI：
10.1016/j.bbagen.2016.12.002
发表时间：
2017-11
期刊：
Biochimica et biophysica acta. General subjects
影响因子：
--
作者：
Gan Q;Fan C
通讯作者：
Fan C
影响因子：
64.8
作者：
Fredens, Julius;Wang, Kaihang;Chin, Jason W.
通讯作者：
Chin, Jason W.
影响因子：
3.5
作者：
George, Susanna;Aguirre, Jacob D.;O'Donoghue, Patrick
通讯作者：
O'Donoghue, Patrick