Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays
Ingrid Remy,Stephen W. Michnick +1 more
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TLDR
The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both, and high-throughput drug screening and quantitative analysis of induction or disruption of protein-protein interactions are also made possible.Abstract:
Two strategies are described for detecting constitutive or induced protein–protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP (FK506 binding protein) and FRAP (FKBP–rapamycin binding protein) fused to DHFR fragments and show a rapamycin dose-dependent survival of clones that requires ≈25 molecules of reconstituted DHFR per cell. A fluorescence assay also is described, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Formation of the FKBP–rapamycin–FRAP complex is detected in stably and transiently transfected cells. Quantitative rapamycin dose-dependence of this complex is shown to be consistent with in vitro binding and distinguishable from a known constitutive interaction of FKBP and FRAP. We also show that this strategy can be applied to study membrane protein receptors, demonstrating dose-dependent activation of the erythropoietin receptor by ligands. The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both. High-throughput drug screening and quantitative analysis of induction or disruption of protein–protein interactions are also made possible.read more
Citations
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Firefly Luciferase Complementation Imaging Assay for Protein-Protein Interactions in Plants
TL;DR: This assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants, and Mutants that are known to compromise protein- protein interactions showed little or much reduced luciferase activity.
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TL;DR: In this article, the authors optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of the firefly fragment.
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