Abstract
We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.
Keywords: Membrane proteins, protein interaction, protein proximity, biosensor, fluorogen-activating protein, tethered fluorogen assay, TEFLA.
Combinatorial Chemistry & High Throughput Screening
Title:Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins
Volume: 19 Issue: 5
Author(s): Kalin V. Vasilev, Eugenio Gallo, Nathaniel Shank and Jonathan W. Jarvik
Affiliation:
Keywords: Membrane proteins, protein interaction, protein proximity, biosensor, fluorogen-activating protein, tethered fluorogen assay, TEFLA.
Abstract: We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.
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Cite this article as:
V. Vasilev Kalin, Gallo Eugenio, Shank Nathaniel and W. Jarvik Jonathan, Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins, Combinatorial Chemistry & High Throughput Screening 2016; 19 (5) . https://dx.doi.org/10.2174/1386207319666160408150320
DOI https://dx.doi.org/10.2174/1386207319666160408150320 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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