Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein–Protein Interactions in Cells



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.


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.


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.


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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Kerppola TK (2006) Complementary methods for studies of protein interactions in living cells. Nat Methods 3:969–971

  2. 2.

    Kerppola TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc 1:1278–1286

  3. 3.

    Ciruela F (2008) Fluorescence-based methods in the study of protein–protein interactions in living cells. Curr Opin Biotech 19:338–343

  4. 4.

    Ghosh I, Hamilton AD, ReganL (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122:5658–5659

  5. 5.

    Michnick SW (2003) Protein fragment complementation strategies for biochemical network mapping. Curr Opin Biotech 14:610–617

  6. 6.

    Paulmurugan R, Gambhir SS (2003) Monitoring protein-protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal Chem 75:1584–1589

  7. 7.

    Hu CD, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9:789–798

  8. 8.

    Luker KE, Smith MC, Luker GD et al (2004) Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA 101:12288–12293

  9. 9.

    Kodama Y, Wada M (2009) Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis. Plant Mol Biol 70:211–217

  10. 10.

    Skarp KP, Zhao X, Weber M, Jantti J (2008) Use of bimolecular fluorescence complementation in yeast Saccharomyces cerevisiae. Methods Mol Biol 457:165–175

  11. 11.

    Hiatt SM, Shyu YJ, Duren HM et al (2008) Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans. Methods 45:185–191

  12. 12.

    Nam KH, Kwon OY, Sugiyama K et al (2007) Structural characterization of the photoswitchable fluorescent protein Dronpa-C62S. Biochem Biophys Res Commun 354:962–967

  13. 13.

    Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306:1370–1373

  14. 14.

    Mizuno H, Mal TK, Walchli M et al (2008) Light-dependent regulation of structure flexibility in a photochromic fluorescent protein. Proc Natl Acad Sci USA 105:9927–9932

  15. 15.

    Aramaki S, Hatta K (2006) Visualizing neurons one-by-one in vivo: optical dissection and reconstruction of neural networks with reversible fluorescent proteins. Dev Dyn 235:2192–2199

  16. 16.

    Venclovas C, Thelen MP (2000) Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic Acids Res 28:2481–2493

  17. 17.

    Zou L, Cortez D, Elledge SJ (2002) Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev 16:198–208

  18. 18.

    Shiomi Y, Shinozaki A, Nakada D et al (2002) Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9-1-1 and Rad17-RFC. Genes Cells 7:861–868

  19. 19.

    Bermudez VP, Lindsey-Boltz LA, Cesare, A.J.; et al. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc. Natl. Acad. Sci. USA 100: 1633-1638, 2003.

  20. 20.

    Bao S, Lu T, Wang X et al (2004) Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene 23:5586–5593

  21. 21.

    Lee J, Kumagai A, Dunphy WG (2007) The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. J Biol Chem 282:28036-28044

  22. 22.

    Gembka A, Toueille M, Smirnova E et al (2007) The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta in long patch base excision repair. Nucleic Acids Res 35:2596–2608

  23. 23.

    Wang W, Brandt P, Rossi ML et al (2004) The human Rad9-Rad1-Hus1 checkpoint complex stimulates flap endonuclease 1. Proc Natl Acad Sci USA 101:16762–16767

  24. 24.

    Friedrich-Heineken E, Toueile M, Tannler B et al (2005) The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially relate flap endonuclease I activity. J Mol Biol 353:980–989

  25. 25.

    Toueille M, El-Andaloussi N, Frouin I et al (2004) The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilization efficiency: implications for DNA repair. Nucleic Acid Res 32:3316–3324

  26. 26.

    Smirnova E, Toueille M, Markkanen E et al (2005) The human checkpoint sensor and alternative DNA clamp Rad9-Rad1-Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery. Biochem J 389:13–17

  27. 27.

    Shi G, Chang DY, Cheng CC et al (2006) Physical and functional interactions between MutY homolog (MYH) and checkpoint proteins Rad9-Rad1-Hus1. Biochem J 400:53–62

  28. 28.

    Guan X, Bai H, Shi G et al (2007) The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates NEIL1 glycosylase. Nucleic Acids Res 35:2463–2472

  29. 29.

    Guan X, Madabushi A, Chang DY et al (2007) The human checkpoint sensor Rad9-Rad1-Hus1 interacts with and stimulates DNA repair enzyme TDG glycosylase. Nucleic Acids Res 35:6207–6218

  30. 30.

    Slupska MM, Baikalov C, Luther WM et al (1996) Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage. J Bacteriol 178:3885–3892

  31. 31.

    Ohtsubo T, Nishioka K, Imaiso Y et al (2000) Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria. Nucleic Acid Res 28:1355–1364

  32. 32.

    Shinmura K, Kasai H, Sasaki A et al (1997) 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) DNA glycosylase and AP lyase activities of hOGG1 protein and their substrate specificity. Mutat Res 385:75–82.

  33. 33.

    Sakumi K, Furuichi M, Tsuzuki T et al (1993) Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis. J Biol Chem 268:23524–23530

  34. 34.

    Parker A, Gu Y, Mahoney W et al (2001) Human homolog of the MutY repair protein (hMYH) physically interacts with proteins involved in long patch DNA base excision repair. J Biol Chem 276:5547–5555

  35. 35.

    Gu Y, Parker A, Wilson TM et al (2002) Human MutY homolog, a DNA glycosylase involved in base excision repair, physically and functionally interacts with mismatch repair proteins human MutS homolog 2/human MutS homolog 6. J Biol Chem 277:11135–11142

  36. 36.

    Chang DY, Lu AL (2005) Interaction of checkpoint proteins Hus1/Rad1/Rad9 with DNA base excision repair enzyme MutY homolog in fission yeast, Schizosaccharomyces pombe. J Biol Chem 280:408–417

  37. 37.

    Lupardus PJ, Cimprich KA (2006) Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of claspin and the Rad9 carboxy terminus. Mol Biol Cell 17:1559–1569

  38. 38.

    Brünger AT, Adams PD, Clore GM et al (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921

  39. 39.

    Campbell RE, Tour O, Palmer AE et al (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882

  40. 40.

    Jach G, Pesch M, Richter K et al (2006) An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat Methods 3:597–600

  41. 41.

    Hirai I, Sasaki T, Wang HG (2004) Human hRad1 but not hRad9 protects hHus1 from ubiquitin-proteosomal degradation. Oncogene 23:5124–5130

  42. 42.

    Doré AS, Kilkenny ML, Rzechorzek NJ, Pearl LH (2009) Crystal structure of the Rad9-Rad1-Hus1 DNA damage checkpoint complex-implications for clamp loading and regulation. Molecular Cell 34:735–745

  43. 43.

    Sohn SY, Cho Y (2009) Crystal structure of the human Rad9-Rad1-Hus1 clamp. J Mol Biol 390:490–502

  44. 44.

    Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21:539–545

  45. 45.

    Chudakov DM, Lukyanov S, Lukyanov KA (2005) Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol 23:605–613

  46. 46.

    Giepmans BN, Adams SR, Ellisman MH et al (2006) The fluorescent toolbox for assessing protein location and function. Science 312:217–224

  47. 47.

    Allen MD, Xhang J (2006) Subcellular dynamics of protein kinase A activity visualized by FRET-based reporter. Biochem Biophys Res Commun 348:716–721

  48. 48.

    Lukyanov KA, Chudakov DM, Lukyanov S et al (2005) Innovation: Photoactivatable fluorescent proteins. Nat Rev Mol Cell Biol 6:885–891

Download references


This work was supported by Real Time Molecular Imaging Project of the Korea Ministry of Science and Technology, National Research Foundation of Korea Grant funded by Korean Government (2009-0074848), Priority Research Centers Program through the National research Foundation of Korea (2009-0093824), and World Class University (WCU, R33-2008-000-1071) program through the Korea Science and Engineering Foundation funded by the Ministry of Education, Science and Technology.

Author information

Correspondence to Ye Sun Han.

Additional information

You Ri Lee and Jong-Hwa Park contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lee, Y.R., Park, J., Hahm, S. et al. Development of Bimolecular Fluorescence Complementation Using Dronpa for Visualization of Protein–Protein Interactions in Cells. Mol Imaging Biol 12, 468–478 (2010). https://doi.org/10.1007/s11307-010-0312-2

Download citation

Key words

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