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Spying on Protein Interactions in Living Cells with Reconstituted Scarlet Light


BiFC (Bimolecular fluorescence complementation) assay and BiFC combining with FRET (Fluorescence resonance energy transfer) technique have become important tools for molecular interaction studies in live cells. However, the real detection and cellular imaging performances of most existing red fluorescent protein-derived BiFC assays still suffer from relative low ensemble brightness, high cytotoxicity, prone-to-aggregation or severe residual dimerization, inefficient complementation and slow maturation at 37℃ physiological temperature in live mammalian cells. We developed a BiFC assay based on a recently evolved truly monomeric red fluorescent protein (FP) mScarlet-I with excellent cellular performances such as low cytotoxicity, fast and efficient chromophore maturation and the highest in-cell brightness among all previously reported monomeric red fluorescent proteins. In this work, classic β-Fos/β-Jun constitutive heterodimerization model and rapamycin-inducible FRB/FKBP interaction system were used to establish and test the performances of mScarlet-I-based BiFC assay in live mammalian cells. Furthermore, simply by adopting large-Stokes-shift fluorescent protein mAmetrine as donor, β-Jun-β-Fos-NFAT1 ternary protein complex formation could be readily and efficiently detected and visualized with minimal spectral cross-talk in live HeLa cell by combining live-cell sensitized-emission FRET measurement with mScarlet-I-based BiFC. The currently established BiFC assay in this work was also shown to be able to detect and visualize various protein-protein interactions (PPIs) at different subcellular compartments with high specificity and sensitivity under 37℃physiological temperature in live mammalian cells.

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Publication details

The article was received on 04 Jul 2018, accepted on 03 Sep 2018 and first published on 03 Sep 2018

Article type: Paper
DOI: 10.1039/C8AN01223G
Citation: Analyst, 2018, Accepted Manuscript
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    Spying on Protein Interactions in Living Cells with Reconstituted Scarlet Light

    S. wang, M. Ding, B. Xue, Y. Hou and Y. Sun, Analyst, 2018, Accepted Manuscript , DOI: 10.1039/C8AN01223G

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