Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
The gold standard for tracking a protein of interest (POI) in cells is to fluorescently label it. Put green fluorescent protein (GFP) in frame with the POI, for example, and when the POI is expressed, voila! It glows.
But GFP and its relatives aren’t perfect. At approximately 240 amino acids, these structures are large enough to potentially disrupt normal protein function and trafficking. And, because they’re fluorescent as soon as they’re folded, fluorescent proteins (FPs) aren’t ideal for studying time- or spatially dependent phenomena.
There are other ways to track a POI that overcome some of these issues and can introduce additional functionalities, as well. Several such systems are commercially available, and others have been published. Most make use of one of three defining modes of action. One relies on an encoded enzymatic moiety that is able to link a substrate to itself, and the second uses a small encoded tag to which an exogenously added enzyme attaches a substrate. The last option uses a distinct, encoded recognition sequence to which a substrate directly reacts.
Self-labeling
Probably the most widely used commercial labeling systems are New England Biolabs’ (NEB’s) SNAP-tag® and Promega’s HaloTag® self-labeling enzyme systems, says Justin Taraska, an investigator at the National Institutes of Health who wrote a review on the topic [1]. These tags are readily available, and the enzymes have been tested and work well in vitro and in vivo, Taraska says.
What self-labeling tags have in common is that a substrate, composed of a constant reactive group coupled to a variable functional group, is recognized and bound by an enzyme linked to a specific POI. For imaging, the functional group is typically an organic fluorophore—fluorescein or hexachlorofluorescein, in the case of ActiveMotif’s LigandLink™, for instance.
SNAP-tag is based on an O6-alkylguanine-DNA alkytransferase (AGT), a human DNA-repair enzyme that recognizes benzylguanine. NEB’s CLIP-tag® uses a modified AGT that recognizes benzylcytosine. HaloTag is derived from a bacterial dehalogenase that recognizes chloroalkane substrates while LigandLink uses E. coli dihydrofolate reductase to recognize the antibiotic trimethoprim.
Though similar in concept, these systems are not necessarily interchangeable. LigandLink is available in both covalent and noncovalent designs, but the others offer only permanent linkages. Fixatives, detergents and other treatments may affect one reaction more than another, and the size of the tag and the label, the spectral properties of the associated fluors and toxicity of the reagents can all impact your experiment.
Each platform sports a unique set of functional groups to which the reactive ligand can be bound. For some, this includes solid surfaces such as beads, slides and plates, to enable protein capture. SNAP-tag, for example, can be derivitized with oligonucleotides, biotin, quantum dots, gold or even a nonfluorescent blocker, says Ivan Corrêa, senior scientist in NEB's division of chemical biology. Customers can build their own applications, as well, going beyond the already functionalized dyes and affinity reagents in the catalog.
Pre-cloned vectors also are often available. For example Promega, in collaboration with Kazusa Institute, has generated a library comprising several thousand HaloTag fusions, says Marjeta Urh, Promega’s director of research.
Enzyme-mediated labeling
NEB markets a second platform for protein labeling, based on the acyl carrier protein (ACP). Here, a small (8 kD) ACP-tag (or MCP-tag) is genetically fused to the POI. The functional group, conjugated with a substrate derived from Coenzyme A (CoA), then can be linked covalently to the tag by means of a recombinant synthase added to the buffer. As with SNAP-tag and CLIP-tag, ACP-tag and MCP-tag can be used in tandem to perform dual labeling.
Unlike the self-labeling enzyme platforms, these tags cannot be used to label proteins in the cytoplasm, because the ACP and MCP enzymes are unable to cross the cell membrane, Corrêa says. “The ACP/MCP tags are very good for labeling the membrane (or for in vitro labeling).”
A host of systems based on sortases, lipoic acid ligases and other ligases and transferases, using similar principles, also have been reported. [2] But “there are very few pieces of work published using those enzyme-mediated tags to label intracellular proteins in context,” Corrêa notes.
Tetracysteine, or, the 4 C’s
The third class of system comprises a genetically encoded, six-amino acid tetracysteine (TC) tag and membrane-permeable biarsenical fluorescent dyes. The dye isn’t fluorescent on its own, as it is complexed with an inhibitory molecule. But in the presence of the TC tag, that inhibitor is ejected, making the dyes light up.
Initially marketed by Invitrogen under the Lumio™ name, kits were re-introduced by Thermo Fisher Scientific (which acquired Life Technologies, which in turn had acquired Invitrogen) as TC-FlAsH™ and TC-ReAsH™. These kits contain British anti-Lewisite (BAL) wash buffer, which “blocks any nonspecific binding of our FlAsH/ReAsH to the TC-like tags,” notes Mike O’Grady, senior R&D manager in the cell-imaging and analysis group at Thermo Fisher Scientific. “It gives you a higher signal-to-noise [ratio], which is what the game is all about, and why GFP is so well liked.”
Applications
Yet unlike GFP, which is always “on,” the tags discussed here don’t fluoresce until they bind a substrate. By varying the timing that dyes are added, using different color dyes (and perhaps blockers) and/or combining membrane-permeable with membrane-impermeable dyes, new experimental possibilites arise that would not be possible with an encoded FP.
Pulse-chase experiments enable one to study protein synthesis and degradation as two separate events. “First, label your protein with one ligand, in one color [and then] remove the ligand from the cell media and add a new ligand to the media in a different color, so all the proteins synthesized after this point are then labeled in a different color,” Urh explains.
Likewise, using a dye that can’t cross the membrane by itself allows the study of receptor internalization, recycling and degradation. Tagging a receptor with an FP would yield signal throughout the cell, “but in the case of SNAP-tag, they only become fluorescent when they react with the substrate on the surface,” points out Corrêa.
Vendors and researchers are constantly improving protein-tagging technology and expanding the offerings—from 12-amino acid TC tags with greater specificity and TC-reactive substrates with less toxicity, to brighter and more stable fluors allowing for ever higher-resolution microscopy. Ligands can be functionalized with reagents for PET or X-ray imaging, with pH-sensitive or photoswitchable dyes or with moieties that tag a protein for destruction. And NEB is about to release the first cell-permeable, near-infrared dye, for use with the SNAP-Tag system.
If you want to know what your POI is doing, get a tag on it!
Reference
[1] Crivat, G, and Taraska, JW, “Imaging proteins inside cells with fluorescent tags,” Trends Biotechnol, 30:8-16, 2012. [PubMed ID: 21924508]
[2] Wang, Z., et al., “Engineered fluorescence tags for in vivo protein labelling,” RSC Adv, 4:7235–45, 2014. [Journal] [PubMed ID: NA]