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

Abstract

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 live cell applications, partly because it needs to be adjusted to the specific protein under study. Here, we present a generic, 14-residues 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, can be substituted with other epitopes and, in some cases, can be completely removed, reducing the tag length to 5 residues. The GCE-tag presented here offers a powerful, easy-to-implement tool for live cell labeling of cellular proteins with small and bright probes. 3 BACKGROUND

Live cell single molecule tracking and localization microscopy of bioorthogonally labeled plasma membrane proteins

“…33,34 To test this possibility, we inserted a 14-residue long peptide linker that encodes for BCNK, which we have recently optimized for biorthogonal labeling (Fig. S1a, † bottom panel), 46 between positions 344 and 345 in Shaker B. Using the linker, specific PM labeling of Shaker B was obtained using any of the dyes tested, suggesting that displacing BCNK from the PM may improve labeling (Fig.…”

Using unnatural amino acids to selectively label proteins for cellular imaging: a cell biologist viewpoint

“…In all the above-mentioned examples, the unAA was incorporated at the extracellular regions of the protein and labeling was performed using cell-impermeable Tet-Fl-dyes, thus avoiding the relatively high background levels associated with the technique (see subsection The click reaction, below). After further optimization, the GCE-based labeling technique was successfully applied for intracellular labeling, and various intracellular components-including intermediate filaments, microtubules, intracellular vesicles, the PM, lysosomes, and the endoplasmic reticulum-were labeled and imaged in live mammalian cells [22,24,[35][36][37][38]. Here, too, small proteins that could not be labeled using protein tags were successfully labeled and imaged.…”

“…Genetic code expansion-based intra-and extracellular labeling was also used in photomanipulation techniques, such as FRET and FRAP [4,30,33,34,36,38] ( Fig. 2A).…”

“…The ratio between the specific labeling of the POI and the unspecific labeling of other proteins in the cell can be quantified by using in-gel fluorescence [22,23,30,36,47]. In addition, the overall background level in cells can be evaluated by measuring the signal intensity after adding all the GCE and click reaction components to the cells-except the TAG-modified POI [22,23,30,35,36,38,47]. Despite these potential background-related limitations, GCEbased labeling demonstrates relatively high signal-tonoise ratios (SNRs), which enable quantitative imaging in either fixed or live cells [24,35,36].…”

“…Tailoring the characteristics of the Fl-dye to the desired imaging application is a major advantage of GCEbased labeling and can greatly improve the performance of advanced imaging techniques. While some dyes are cell-permeable, others are not; some dyes may be more suited for labeling certain cellular components; and while some dyes are bright and photostable, others exhibit blinking properties, which are required in SMLM experiments [19,36]. The cellular permeability and fluorogenicity of various Tet-Fl-dyes were recently reported and can be used as a guideline for a rational selection of the Fl-dye [13,24].…”