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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Citations (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.

Original languageEnglish
Article number350
JournalCommunications Biology
Issue number1
Publication statusPublished - 2020 Dec 1

ASJC Scopus subject areas

  • Medicine (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)


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