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
- 이유리; 박종화; 함수현; 강린우; 정지형; 남기현; 황광현; 권익찬; 한예선
- 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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