title: An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes.
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-tRNATrppair 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-tRNATrpvariants 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.
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: James S
authors.identifier: 0000-0003-2653-5516
authors.initials: JS
authors.lastname: Italia
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Partha Sarathi
authors.initials: PS
authors.lastname: Addy
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Chester J J
authors.initials: CJ
authors.lastname: Wrobel
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Crawford
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington, USA.
authors.forename: Marc J
authors.identifier: 0000-0002-0477-5511
authors.initials: MJ
authors.lastname: Lajoie
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Yunan
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Chemistry, Boston College, Chestnut Hill, Massachusetts, USA.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Chatterjee
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D056890:Eukaryota / Q000235:genetics*; D005815:Genetic Code / Q000235:genetics*; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D008968:Molecular Conformation; D012343:RNA, Transfer / Q000235:genetics* / Q000378:metabolism; D014369:Tryptophan-tRNA Ligase / Q000378:metabolism*
chemical_list: D012343:RNA, Transfer; D014369:Tryptophan-tRNA Ligase