Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
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Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved April 28, 2010 (received for review December 7, 2009)
↵1C.U., K.A.W., and H.B. contributed equally to this work.
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.
Footnotes
- 2To whom correspondence should be addressed. E-mail: ating{at}mit.edu.
Author contributions: C.U., K.A.W., H.B., S.T., M.F.-S., S.P., and A.Y.T. designed research; C.U., K.A.W., H.B., and S.T. performed research; C.U., K.A.W., H.B., S.T., and A.Y.T. analyzed data; and C.U., K.A.W., and A.Y.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914067107/-/DCSupplemental.
A fluorophore ligase for site-specific protein labeling inside living cells
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