Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

Keita Hibi, Kazuaki Amikura, Naoki Sugiura, Keiko Masuda, Satoshi Ohno, Takashi Yokogawa, Takuya Ueda, Yoshihiro Shimizu*


研究成果: Article査読

29 被引用数 (Scopus)


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.

ジャーナルCommunications Biology
出版ステータスPublished - 2020 12月 1

ASJC Scopus subject areas

  • 医学(その他)
  • 生化学、遺伝学、分子生物学(全般)
  • 農業および生物科学(全般)


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