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Development of bimolecular fluorescence complementation using Dronpa for visualization of protein-protein interactions in cells

Authors
 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 
Citation
 MOLECULAR IMAGING AND BIOLOGY, Vol.12(5) : 468-478, 2010 
Journal Title
MOLECULAR IMAGING AND BIOLOGY
ISSN
 1536-1632 
Issue Date
2010
MeSH
Amino Acid Sequence ; Blotting, Western ; Cell Line ; Fluorescence ; Humans ; Immunoprecipitation ; Molecular Sequence Data ; Protein Binding ; Proteins/chemistry ; Proteins/metabolism* ; Sequence Homology, Amino Acid
Keywords
Bimolecular fluorescence complementation ; Dronpa ; Reversible photoswitching activity ; Protein–protein interaction ; Human MutY homolog ; hHus1 ; hRad1
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
Full Text
http://link.springer.com/article/10.1007%2Fs11307-010-0312-2
DOI
10.1007/s11307-010-0312-2
Appears in Collections:
1. College of Medicine (의과대학) > Research Institute (부설연구소) > 1. Journal Papers
Yonsei Authors
Chung, Ji Hyung(정지형)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/102516
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