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

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

Research output: Contribution to journalArticle

24 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)468-478
Number of pages11
JournalMolecular Imaging and Biology
Volume12
Issue number5
DOIs
Publication statusPublished - 2010 Oct

Keywords

  • Bimolecular fluorescence complementation
  • Dronpa
  • Human MutY homolog
  • Protein-protein interaction
  • Reversible photoswitching activity
  • hHus1
  • hRad1

ASJC Scopus subject areas

  • Oncology
  • Radiology Nuclear Medicine and imaging
  • Cancer Research

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