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

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.

journal_name

Mol Imaging Biol

journal_title

Molecular imaging and biology

authors

Lee YR,Park JH,Hahm SH,Kang LW,Chung JH,Nam KH,Hwang KY,Kwon IC,Han YS

doi

10.1007/s11307-010-0312-2

subject

Has Abstract

pub_date

2010-10-01 00:00:00

pages

468-78

issue

eissn

1536-1632

issn

1860-2002

journal_volume

pub_type

杂志文章

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