GFP-complementation assay to detect functional CPP and protein delivery into living cells

Abstract

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease.

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.…”

Nanoparticle mediated delivery and small molecule triggered activation of proteins in the nucleus

“…Recently, GFP-based bimolecular fluorescence complementation (BiFC) assays have been developed, consisting of two proteins derived from genetic splitting of a fluorescent protein that only fluoresce after complementation. BiFC assays have been successfully employed for the comparison of cell penetrating peptide (CPP) function and the assessment of antibody internalization[30–32]. They deliver a relative fluorescent readout which is ideal for optimization processes.…”