Abstract
BackgroundIn the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. This labeling approach is currently not broadly used in live-cell applications, partly because it needs to be adjusted to the specific protein under study.ResultsWe present a generic, 14-residue long, N-terminal tag for GCE-based labeling of proteins in live mammalian cells. Using this tag, we generated a library of GCE-based organelle markers, demonstrating the applicability of the tag for labeling a plethora of proteins and organelles. Finally, we show that the HA epitope, used as a backbone in our tag, may be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues.ConclusionsThe GCE-tag presented here offers a powerful, easy-to-implement tool for live-cell labeling of cellular proteins with small and bright probes.
Highlights
In the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes
* Correspondence: elianat@post.bgu.ac.il 1Department of Life Sciences, Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel 2National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, 84105 Beer Sheva, Israel Full list of author information is available at the end of the article In GCE-based labeling, a non-canonical amino acid carrying a functional group is incorporated into the sequence of a protein in response to an in-frame amber stop codon (TAG), via an orthogonal tRNA/tRNA-synthetase pair
For GCE-based labeling, we chose to design an N-terminal tag in order to avoid protein truncations resulting from inefficient incorporation of the non-canonical amino acid (ncAA) [5, 6, 16, 17]
Summary
In the high-resolution microscopy era, genetic code expansion (GCE)-based bioorthogonal labeling offers an elegant way for direct labeling of proteins in live cells with fluorescent dyes. Using genetic code expansion (GCE) and bioorthogonal chemistry, it is possible to attach fluorescent dyes (Fl-dyes) to specific protein residues, thereby allowing direct labeling of proteins in live cells with Fl-dyes [1,2,3] This approach has been applied, in recent years, for fluorescent labeling of extra- and intracellular proteins [4,5,6,7,8,9,10]. Successful labeling relies on the exogenous expression of an orthogonal tRNA/tRNA-synthetase pair and a protein of interest (bearing a ncAA) at sufficient levels to allow effective labeling
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