1. Development of bimolecular fluorescence complementation using Dronpa for visualization of protein-protein interactions in cells.
- Author
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You Ri Lee, Jong-Hwa Park, Soo-Hyun Hahm, Lin-Woo Kang, Ji Hyung Chung, Ki-Hyun Nam, Kwang Yeon Hwang, Ick Chan Kwon, Ye Sun Han, Lee, You Ri, Park, Jong-Hwa, Hahm, Soo-Hyun, Kang, Lin-Woo, Chung, Ji Hyung, Nam, Ki-Hyun, Hwang, Kwang Yeon, Kwon, Ick Chan, and Han, Ye Sun
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PROTEIN-protein interactions , *FLUORESCENCE microscopy , *COMPLEMENTATION (Genetics) , *CELLS , *PROTEIN analysis , *PROTEIN metabolism , *AMINO acids , *CELL lines , *COMPARATIVE studies , *DOCUMENTATION , *LIGHT , *RESEARCH methodology , *MEDICAL cooperation , *PROTEINS , *RESEARCH , *WESTERN immunoblotting , *EVALUATION research , *PRECIPITIN tests - Abstract
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. [ABSTRACT FROM AUTHOR]- Published
- 2010
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