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一个无细胞基因表达平台,用于发现和特征化停止子抑制tRNAs

Kosuke Seki^1,2,3, Joey L Galindo^1,2,3, Ashty S Karim^1,2,3

¹Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208, United States.

ACS chemical biology

|May 31, 2023

概括

这项研究引入了一种快速的无细胞系统,用于发现和表征抑制性tRNA,这对于将非正规氨基酸 (ncAA) 纳入蛋白质至关重要. 这种方法加速了用于合成生物学应用的新型直角翻译系统的开发.

科学领域:

合成生物学合成生物学
分子生物学分子生物学
生物化学生物化学

背景情况:

非正规氨基酸 (ncAAs) 能够使蛋白质具有新的特性和功能.
特定站点的ncAA整合依赖于直角翻译系统 (OTS).
目前用于发现抑制器tRNA的方法是费力和耗时的.

研究的目的:

开发一种快速的无细胞基因表达系统,用于抑制器tRNAs的功能特征.
展示该系统在发现新型抑制性tRNA和实现多重 ncAA 结合方面的实用性.

主要方法:

使用以大肠杆菌原始提取物为基础的无细胞系统.
与超叠绿色光蛋白 (sfGFP) 记者同时表达的正交tRNAs,其中包含一个停止编码子.
评估了UAG和UAA抑制活性,并证明了两种正交tRNA的共同转录,用于双重ncAA整合.

主要成果:

在无细胞系统中成功表达和特征功能抑制器tRNAs.
在单一蛋白质中证明了多重的ncAA合并.
从使用无细胞工作流程的元基因组数据中确定了假定的UAG抑制器tRNA.

结论:

开发的无细胞系统显著加速了功能抑制器tRNAs的发现和表征.
这个平台促进了合成生物学直角翻译系统的快速扩展.
通过ncAA的整合,使蛋白质具有新型功能的高效工程.

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