Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency.
Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency.
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DOI：
10.1002/cbic.201700039
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发表时间：
2017-06-19
期刊：
影响因子：
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通讯作者：
Fasan R
中科院分区：
文献类型：
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作者：
Owens AE;Grasso KT;Ziegler CA;Fasan R
通过琥珀终止抑制的遗传密码扩增提供了一个强大的工具，用于将非蛋白质功能引入蛋白质中，用于广泛的应用。然而，与有义密码子翻译相比，通过工程改造的阿尔斯进行的非典型氨基酸（ncAA）的核糖体掺入通常以显著降低的效率进行。在这里，我们报告了一个通用的平台，用于开发工程氨酰-tRNA合成酶，其通过琥珀终止密码子抑制介导ncAA掺入的效率提高。该系统将白色/蓝色菌落筛选与基于平板的比色测定相结合，从而将高通量能力与AARS依赖性ncAA掺入效率的可靠和定量测量相结合。该双层功能筛选系统成功地应用于获得吡咯赖氨酰-tRNA合成酶变体（CrtK-RS（4.1）），其具有显著提高的效率（+250-370%），以介导Nε-巴豆酰-赖氨酸和与蛋白质翻译后修饰研究相关的其他赖氨酸类似物掺入靶蛋白中。有趣的是，发现由CrtK-RS（4.1）积累的有益突变定位在酶的非催化N-末端结构域内，并且可以转移到另一个PylRS变体，从而提高其掺入其相应ncAA底物的能力。这项工作引入并验证了一个有效的平台，用于改善AARS，可以很容易地扩展到该酶家族的其他成员和/或其他目标非经典氨基酸。开发了一种涉及白色/蓝色菌落筛选与基于平板的功能测定偶联的双层筛选系统，并将其应用于进化吡咯赖氨酰-tRNA合成酶变体，其具有提高的效率，朝向通过琥珀终止密码子抑制将巴豆酰赖氨酸和其他赖氨酸衍生的非规范氨基酸掺入蛋白质中
Genetic code expansion via amber stop suppression provides a powerful tool for introducing non-proteinogenic functionalities into proteins for a broad range of applications. However, ribosomal incorporation of non-canonical amino acids (ncAA) by means of engineered AARS often proceeds with significantly reduced efficiency compared to sense codon translation. Here, we report the implementation of a versatile platform for the development of engineered aminoacyl-tRNA synthetases with enhanced efficiency in mediating ncAA incorporation via amber stop codon suppression. This system integrates a white/blue colony screen with a plate-based colorimetric assay, thereby combining high-throughput capabilities with reliable and quantitative measurement of AARS-dependent ncAA incorporation efficiency. This two-tier functional screening system was successfully applied to obtain a pyrrolysyl-tRNA synthetase variant (CrtK-RS(4.1)) with significantly improved efficiency (+250-370%) toward mediating the incorporation of Nε-crotonyl-lysine and other lysine analogs of relevance for the study of protein post-translational modifications, into a target protein. Interestingly, the beneficial mutations accumulated by CrtK-RS(4.1) were found to localize within the non-catalytic N-terminal domain of the enzyme, and could be transferred to another PylRS variant improving its ability to incorporate its corresponding ncAA substrate. This work introduces and validates an efficient platform for the improvement of AARSs that could be readily extended to other members of this enzyme family and/or other target non-canonical amino acids. A two-tier screening system involving a white/blue colony screen coupled to a plate-based functional assay was developed and applied to evolve pyrrolysyl-tRNA synthetase variants with improved efficiency toward the incorporation of crotonyl lysine and other lysine-derived non-canonical amino acids into proteins via amber stop codon suppression