Some of these applications 8, 9, 10 fused the small GFP11 fragment to the target protein as an epitope tag. This split GFP system has been previously used for protein quantification 5, visualization of protein subcellular localization 8, 9, 10, single-molecule imaging 11, cell-cell contact detection 12, as well as in vitro protein complex assembly 13. Upon complementation, the reconstituted GFP becomes fluorescent after the chromophore maturation reaction is completed 5, 7. The GFP1-10 fragment, which contains the three residues that constitute the GFP chromophore, is nonfluorescent by itself because chromophore maturation requires the conserved E222 residue located on GFP11 (ref. This split construct breaks the sequence of sfGFP between the tenth and the eleventh β-strand into two parts: GFP1-10 and GFP11, with GFP11 being a short, 16 amino acid peptide ( Fig.
For this purpose, we adopted a split super-folder GFP (sfGFP) that was previously engineered for efficient self-complementation without the assistance of other protein–protein interactions 5. In this case, as long as the binding module is expressed in excess of the target protein, the equilibrium can be shifted towards complete labelling. One approach to relax this affinity and stoichiometry requirement is to have a binding module that becomes fluorescent only when it is on the epitope. Alternatively, the SunTag could have used a higher affinity and a more precise expression stoichiometry between them, so that there is neither incomplete labelling nor background from excessive binding modules. This arrangement enables it to successfully overcome an intrinsic problem such as the low affinity between the tagged protein and the binding module. For example, the SunTag 2 allows a dramatic enhancement of fluorescence signal by bringing as many as 24 green fluorescent proteins (GFPs) to a single target protein. In addition, small epitope tags can be arranged into a multimerization scaffold. A small peptide epitope tag may induce less perturbation than a much larger fluorescent protein. It is highly modular because the functional units can be easily replaced 2. This two-component labelling approach has a number of advantages. Alternatively, it can bind an intracellularly expressed antibody/nanobody to bring in functional protein units, such as a fluorescent protein for imaging 2, 3 or ubiquitin ligase for protein degradation 4. An epitope tag fused to the target protein can become an enzyme substrate to be ligated to a small molecule 1.
These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.Ĭomplementary to direct fluorescent protein fusion, more and more examples of live cell protein labelling and imaging have recently emerged using epitope tags: peptides that do not function by themselves but can be recognized by other intracellularly expressed proteins. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively.
To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The small size of these tags may reduce functional perturbation and enable signal amplification. In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags.
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