Alternatives for fluorescent proteins in live cell imaging

Most people use GFP or other fluorescent proteins (BFP, CFP, YFP, RFP) to tag their protein of interest for live cell imaging. In many cases this works fine. However, occasionally adding a fluorescent protein on the N- or C- terminal causes disruption of the function or localization of the tagged protein (see: Mahen et al. (2014) Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells. Mol. Biol. Cell. E14-06-1091). Recently new ways to tag proteins for live cell imaging have come available.

Late 2014 Tanenbaum et al. published their Sun-tag system in Cell 159: 635-646. It still uses GFP but in this system the protein of interest is tagged with a tiny peptide epitope, GNC4. The protein is coexpressed with GFP-tagged single-chain antibodies (scFv) that recognize the epitope and bind amplifying the signal.

A company called Covalys, now part of NEB, introduced the ACP-tag/MCP-tag for cell surface labelling and a SNAP-tag system to study proteins in live cells. Invitrogen, now part of Life Technologies, has brought us FlAsH and ReAsH.

Both the ACP-tag and MCP-tag are 77aa (GFP = 238 aa) and are based on the Acyl Carrier Protein (ACP) from E. coli. MCP-tag contains two mutations compared with ACP to make it more substrate specific. Both tags can be detected using one of several CoA-based labelling substrates dependent on the excitation you require. The SNAP-tag is a highly engineered version of the human form of O6- alkylguanine-DNA-alkyltransferase (AGT) of around 150 aa. Covalys has released a whole range of para-substituted benzyl guanines bound to different fluorochromes for detection of the SNAP-tag. See also: Bosch et al. (2014) Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells. Biophysical Journal 107: 803–814. 

Invitrogen's FiAsH and ReAsH uses a 6 aa tag (Cys-Cys-Pro-Gly-Cys-Cys). FlAsH is derived from fluorescein and ReAsH from resorufin. They are virtually non-fluorescent when bound to ethane dithiol (EDT). When FlAsH-EDT or ReAsH-EDT bind to tetracysteine (TC) sequences, EDT is displaced and the tags become highly fluorescent in green or red, respectively.

Besides the smaller tag these systems make it also easier to adapt your protocol when combined with fluorescent proteins or antibodies. By changing the substrate you change the colour used to detect your protein without changing the tag on your protein.

Lai et al. published (PNAS USA 112(10):2948-53) the use of HIS-tagged proteins for live cell imaging using a membrane permeable blue emitting fluorescent probe. In their appendix they also show some results of a green emitting variant.

Promega uses a two-step approach in their Halo labelling system. First, a 33 kDa HaloTag is genetically fused to the protein of interest followed by the binding of a application-specific HaloTag ligand. This binding is rapid and irreversible. The ligand can be used for protein purification, protein interaction analysis, and live cell imaging studies. See England et al. 2015.

If you have tried one of these systems, please let me know your experience (good or bad).

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