Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs.
Journal
Communications biology
ISSN: 2399-3642
Titre abrégé: Commun Biol
Pays: England
ID NLM: 101719179
Informations de publication
Date de publication:
03 07 2020
03 07 2020
Historique:
received:
30
04
2020
accepted:
12
06
2020
entrez:
5
7
2020
pubmed:
6
7
2020
medline:
16
6
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 32620935
doi: 10.1038/s42003-020-1074-2
pii: 10.1038/s42003-020-1074-2
pmc: PMC7334211
doi:
Substances chimiques
Amino Acids
0
Proteins
0
RNA, Transfer
9014-25-9
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
350Subventions
Organisme : NIGMS NIH HHS
ID : R35 GM122560
Pays : United States
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