fluorophore ligase for site-specific protein labeling inside living cells

• Published in: Proceedings of the National Academy of Sciences - PNAS 2010-06, Vol.107 (24), p.10914-10919
• Main Authors: Uttamapinant, Chayasith, White, Katharine A, Baruah, Hemanta, Thompson, Samuel, Fernández-Suárez, Marta, Puthenveetil, Sujiet, Ting, Alice Y
• Format: Article
• Language: English
• Subjects: Actin; Actins; Actins - chemistry; Actins - metabolism; Animals; Binding Sites; Biochemistry; Biological Sciences; Catalytic Domain; Cell Line; Cell lines; Cell nucleus; Cells; Cercopithecus aethiops; Chromatography, High Pressure Liquid; COS Cells; Coumarins; E coli; Enzymes; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fluorescence; Fluorescent Dyes - metabolism; Fluorescent indicators; fluorophores; Fusion protein; Green fluorescent protein; HeLa Cells; Humans; imaging; Kinetics; Ligases - chemistry; Ligases - genetics; Ligases - metabolism; Ligation; Lipoic acid; Microscopy; Models, Molecular; Mutagenesis; Mutagenesis, Site-Directed; Neurons; Physical Sciences; Protein Conformation; Proteins; Proteins - chemistry; Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Umbelliferones - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0914067107

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.