A fluorophore ligase for site-specific protein labeling inside living cells
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Ting_A fluorophore.pdf
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1.44 MB
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Author(s)
Uttamapinant, Chayasith
White, Katherine A.
Baruah, Hemanta
Thompson, Samuel
Fernandez-Suarez, Marta
Puthenveetil, Sujiet
Ting, Alice Y.
Date Issued
June 2010
Journal
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences (U.S.)
Citation
Uttamapinant, C., K. A. White, H. Baruah, S. Thompson, M. Fernandez-Suarez, S. Puthenveetil, and A. Y. Ting. “A fluorophore ligase for site-specific protein labeling inside living cells.” Proceedings of the National Academy of Sciences 107, no. 24 (June 15, 2010): 10914-10919.
Version
Final published version
Abstract
Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.
MIT Department
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1073/pnas.0914067107
