Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

在无细胞系统中完整重组tRNA以生产活性蛋白为遗传密码工程带来了灵活性。它还可以为无细胞合成生物学领域做出贡献，该领域旨在构建可自我复制的人造细胞。本文中，我们基于重组的无细胞蛋白质合成（PURE）系统开发了仅配备体外转录tRNA（iVTtRNA）的系统。该系统由21种iVTtRNA组成，没有核苷酸修饰，能够根据重新设计的遗传密码合成活性蛋白。在系统中操纵iVTtRNA成分可实现遗传密码重写。已证明将修饰的核苷酸引入特定的iVTtRNA中对于蛋白质产量和解码保真度都是有效的，在30°C下，DHFR的产量达到了与天然tRNA反应的40％。所开发的系统将证明对研究解码过程很有用，并可用于遗传密码和蛋白质工程应用。