title: Concerted, rapid, quantitative, and site-specific dual labeling of proteins.
abstract: Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Amit
authors.initials: A
authors.lastname: Sachdeva
authors.forename: Kaihang
authors.initials: K
authors.lastname: Wang
authors.forename: Thomas
authors.initials: T
authors.lastname: Elliott
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D001386:Azides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D013696:Temperature
chemical_list: D000596:Amino Acids; D001386:Azides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011994:Recombinant Proteins