{Reference Type}: Journal Article
{Title}: Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set.
{Author}: Fujino T;Sonoda R;Higashinagata T;Mishiro-Sato E;Kano K;Murakami H;
{Journal}: Nat Commun
{Volume}: 15
{Issue}: 1
{Year}: 2024 May 16
{Factor}: 17.694
{DOI}: 10.1038/s41467-024-48056-z
{Abstract}: 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.