A fluorophore ligase for site-specific protein labeling inside living cells
Author(s)
Uttamapinant, Chayasith; White, Katherine A.; Baruah, Hemanta; Thompson, Samuel; Fernandez-Suarez, Marta; Puthenveetil, Sujiet; Ting, Alice Y.; ... Show more Show less
DownloadTing_A fluorophore.pdf (1.437Mb)
PUBLISHER_POLICY
Publisher Policy
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Terms of use
Metadata
Show full item recordAbstract
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.
Date issued
2010-06Department
Massachusetts Institute of Technology. Department of ChemistryJournal
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
ISSN
0027-8424
1091-6490