Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

• Published in: Communications biology Vol. 3; no. 1; p. 350
• Main Authors: Hibi, Keita, Amikura, Kazuaki, Sugiura, Naoki, Masuda, Keiko, Ohno, Satoshi, Yokogawa, Takashi, Ueda, Takuya, Shimizu, Yoshihiro
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 03.07.2020; Nature Publishing Group
• Subjects: 631/45/500; 631/553/552; 82/80; 82/83; Biology; Biomedical and Life Sciences; Cell-free system; Dihydrofolate reductase; Genetic code; Genetic engineering; Life Sciences; Nucleotides; Protein biosynthesis; Protein engineering; Protein structure; Protein synthesis; Proteins; tRNA
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-020-1074-2

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. Keita Hibi et al. develop a system to reconstitute cell-free protein synthesis using only in vitro transcribed tRNA (iVTtRNAs). They use 21 iVTtRNAs with and without nucleotide modifications to successfully synthesize functional proteins with about 40% production yield. Their system will be useful to study gene and protein engineering.