TY - JOUR
T1 - Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs
AU - Hibi, Keita
AU - Amikura, Kazuaki
AU - Sugiura, Naoki
AU - Masuda, Keiko
AU - Ohno, Satoshi
AU - Yokogawa, Takashi
AU - Ueda, Takuya
AU - Shimizu, Yoshihiro
N1 - Funding Information:
We thank Nono Takeuchi-Tomita for helpful discussions. KA was partly supported by an NIGMS grant (R35GM122560 to Prof. Dieter Söll in Yale University). This work was supported by a Grant-in-Aid to KH (19J12955), KA (15K16083), and YS (17H05680) from the Japan Society for the Promotion of Science (JSPS), the Human Frontier Science Program (RGP0043/2017 to YS), the Astrobiology Center Project of the National Institutes of Natural Sciences (AB271004 and AB281007 to KA, and AB311005 to YS and KA), and an intramural Grant-in-Aid from the RIKEN Center for Biosystems Dynamics Research (to YS).
Publisher Copyright:
© 2020, The Author(s).
PY - 2020/12/1
Y1 - 2020/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s42003-020-1074-2
DO - 10.1038/s42003-020-1074-2
M3 - Article
C2 - 32620935
AN - SCOPUS:85087383574
SN - 2399-3642
VL - 3
JO - Communications Biology
JF - Communications Biology
IS - 1
M1 - 350
ER -