title: Genetic encoding of unnatural amino acids for labeling proteins.
abstract: The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labeling proteins with any probe. Here we describe the genetic encoding of dienophile-bearing unnatural amino acids into proteins expressed in Escherichia coli and mammalian cells using the pyrrolysyl-tRNA synthetase/tRNACUA pair and its variants. We describe the rapid fluorogenic labeling of proteins containing these unnatural amino acids in vitro, in E. coli, and in live mammalian cells with tetrazine-fluorophore conjugates in a bioorthogonal Diels-Alder reaction with inverse electron demand. These approaches have been extended to site-specific protein labeling in animals, and we anticipate that they will have a broad impact on the labeling and imaging field.
authors.affiliation: Faculty of Chemistry, Technische Universität München, Institute for Advanced Study, Lichtenbergstr. 4, Garching, 85748, Germany, kathrin.lang@tum.de.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lang
authors.forename: Lloyd
authors.initials: L
authors.lastname: Davis
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D061565:Cycloaddition Reaction; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D015202:Protein Engineering; D020543:Proteome / Q000096:biosynthesis / Q000737:chemistry; D013194:Staining and Labeling
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D020543:Proteome