Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range

Abstract

Transfection of cells with aplasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations.C ytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence.A ni ncrease in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as af usion protein with mCherry.

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

“…These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes. 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 .…”

“…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.…”

“…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.…”