%0 Journal Article
%T Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set.
%A Fujino T
%A Sonoda R
%A Higashinagata T
%A Mishiro-Sato E
%A Kano K
%A Murakami H
%J Nat Commun
%V 15
%N 1
%D 2024 May 16
%M 38755134
%F 17.694
%R 10.1038/s41467-024-48056-z
%X The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors.