| dc.description.abstract |
In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl–tRNA synthetase and tRNA (TrpRS–tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli–optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS–tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS–tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl–tRNA synthetase (aaRS)–tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS–tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes. |
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