Versatile protein tagging in cells with split fluorescent protein

Abstract

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. 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. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. 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.

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

“…By fusing one fragment on a target protein and detecting its association with the other fragment, these constructs have demonstrated powerful applications in the visualization of subcellular protein localization [1][2][3] , quantification of protein aggregation 4 , detection of cytosolic peptide delivery 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3,9,10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .…”

“…With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP [1][2][3][4][5][6][7][8][9][10] in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly.…”

“…Previously, a sfCherry 1-10/11 system 3 was derived from super-folder Cherry, an mCherry variant optimized for folding efficiency 13 . However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.…”

“…2 Protein labeling through transient expression or endogenous knock-in using mNG2 11 . a Fluorescence images of HeLa cells co-expressing either mNG2 11 or GFP 11 labeled H2B or CLTA with the corresponding FP [1][2][3][4][5][6][7][8][9][10] . Scale bars are 10 μm.…”

“…Scale bars are 10 μm. b FACS backgroud from wild-type HEK 293T cells, or HEK 293T cells stably expressing GFP [1][2][3][4][5][6][7][8][9][10] (using the stong SFFV promoter or the weak PGK promoter) or mNG2 [1][2][3][4][5][6][7][8][9][10] (using SFFV). c Comparison of tagging endogenous LMNA, RAB11A, and CLTA using mNG2 11 or GFP 11 knock-in in SFFV-mNG2 [1][2][3][4][5][6][7][8][9][10] and PGK-GFP 1-10 cells, respectively, (flow cytometry histograms) and the corresponding confocal images of mNG2 11 knock-in cells.…”