A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

Abstract

Background: 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. 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. Results: We 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. Conclusions: The GCE-tag presented here offers a powerful, easy-to-implement tool for live-cell labeling of cellular proteins with small and bright probes.