Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein-Protein Interactions in Cells

Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein-Protein Interactions in Cells
Bimolecular fluorescence complementation; Dronpa; Reversible photoswitching activity; Protein?rotein interaction; Human MutY homolog; hHus1; hRad1
Issue Date
Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging
VOL 12, NO 5, 468-478
Purpose: We developed a bimolecular fluorescence complementation (BiFC) strategy using Dronpa, a new fluorescent protein with reversible photoswitching activity and fast responsibility to light, to monitor protein–protein interactions in cells. Procedures: Dronpa was split at residue Glu164 in order to generate two Dronpa fragments [Dronpa N-terminal: DN (Met1–Glu164), Dronpa C-terminal: DC (Gly165–Lys224)]. DN or DC was separately fused with C terminus of hHus1 or N terminus of hRad1. Flexible linker [(GGGGS)×2] was introduced to enhance Dronpa complementation by hHus1–hRad1 interaction. Furthermore, we developed expression vectors to visualize the interaction between hMYH and hHus1. Gene fragments corresponding to the coding regions of hMYH and hHus1 were N-terminally or C-terminally fused with DN and DC coding region. Results: Complemented Dronpa fluorescence was only observed in HEK293 cells cotransfected with hHus1–LDN and DCL–hRad1 expression vectors, but not with hHus1–LDN or DCL–hRad1 expression vector alone. Western blot analysis of immunoprecipitated samples using anti-c-myc or anti-flag showed that DN-fused hHus1 interacted with DC-fused hRad1. Complemented Dronpa fluorescence was also observed in cells cotransfected with hMYH–LDN and DCL– hHus1 expression vectors or hMYH–LDN and hHus1–LDC expression vectors. Furthermore, complemented Dronpa, induced by the interaction between hMYH–LDN and DCL–hHus1, showed almost identical photoswitching activity as that of native Dronpa. Conclusion: These results demonstrate that BiFC using Dronpa can be successfully used to investigate protein–protein interaction in live cells. Furthermore, the fact that complemented Dronpa has a reversible photoswitching activity suggests that it can be used as a tool for tracking protein–protein interaction.
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