title: Expanding the genetic code of Escherichia coli with phosphotyrosine.
abstract: Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to cotranslationally incorporate O-phosphotyrosine into the superfolder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA.|Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
authors.forename: Chenguang
authors.identifier: 0000-0003-0662-1185
authors.initials: C
authors.lastname: Fan
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
authors.forename: Kevan
authors.initials: K
authors.lastname: Ip
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.|Department of Chemistry, Yale University, New Haven, CT, USA.
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D017353:Gene Deletion; D005815:Genetic Code; D049452:Green Fluorescent Proteins / Q000378:metabolism; D063747:Methanocaldococcus / Q000201:enzymology; D010444:Peptide Elongation Factor Tu / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational / Q000235:genetics
chemical_list: D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D010744:Phosphoric Monoester Hydrolases; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases
keywords: aminoacyl-tRNA synthetase; elongation factor; genetic code expansion; phosphatase; phosphotyrosine; tyrosine phosphorylation