Concerted, Rapid, Quantitative, and Site-Specific Dual Labeling of Proteins

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 compl...

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Bibliographic Details
Published inJournal of the American Chemical Society Vol. 136; no. 22; pp. 7785 - 7788
Main Authors Sachdeva, Amit, Wang, Kaihang, Elliott, Thomas, Chin, Jason W
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 04.06.2014
Subjects
Amino Acids - chemistry
Amino Acids - genetics
Azides - chemistry
Communication
Escherichia coli - genetics
Escherichia coli - metabolism
Fluorescent Dyes
Hydrogen-Ion Concentration
Indicators and Reagents
Kinetics
Models, Molecular
Protein Conformation
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Temperature
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Summary: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.
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ISSN:0002-7863
1520-5126
DOI:10.1021/ja4129789