Detection of Protein-Protein Interactions in Live Cells and Animals with Split Firefly Luciferase Protein Fragment Complementation

  • Victor Villalobos
  • Snehal Naik
  • David Piwnica-Worms
Part of the Methods in Molecular Biology™ book series (MIMB, volume 439)


Protein fragment complementation has emerged as a powerful tool for measuring protein-protein interactions in the context of live cells. The adaptation of this strategy for use with firefly luciferase now allows for the non-invasive, quantitative, real-time readout of protein interactions in lysates, live cells, and whole animals. Bioluminescence provides a robust imaging modality due to its extremely low background signal and large dynamic range. The split luciferase fusion constructs described here are inducible by addition of ligands, small molecules or drugs, in this example, rapamycin, and have been shown to work in vivo.


protein fragment complementation protein-protein interactions firefly luciferase split luciferase complementation bioluminescence molecular imaging 



Special thanks to colleagues at the Molecular Imaging Center for valuable discussions. This educational project was supported by NIH grant P50 CA94056.


  1. 1.
    1. Toby G, Golemis E (2001) Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions. Methods 24:201–217PubMedCrossRefGoogle Scholar
  2. 2.
    2. Luker K, Piwnica-Worms D (2004) Optimizing luciferase protein fragment complementation for bioluminescent imaging of protein-protein interactions in live cells and animals. Methods Enzymol 385:349–360PubMedCrossRefGoogle Scholar
  3. 3.
    3. Rossi F, Charlton C, Blau H (1997) Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc Natl Acad Sci USA 94:8405–8410PubMedCrossRefGoogle Scholar
  4. 4.
    4. Wehrman T, Kleaveland B, Her JH, Balint RF, Blau HM (2002) Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments. Proc Natl Acad Sci USA 99:3469–3474PubMedCrossRefGoogle Scholar
  5. 5.
    5. Remy I, Michnick S (1999) Clonal selection and in vivo quantitation of protein interactions with protein-fragment complementation assays. Proc Natl Acad Sci USA 96:5394–5399PubMedCrossRefGoogle Scholar
  6. 6.
    6. Remy I, Wilson I, Michnick S (1999) Erythropoietin receptor activation by a ligand-induced conformation change. Science 283:990–993PubMedCrossRefGoogle Scholar
  7. 7.
    7. Galarneau A, Primeau M, Trudeau L-E, Michnick S (2002) b-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein-protein interactions. Nat Biotechnol 20:619–622PubMedCrossRefGoogle Scholar
  8. 8.
    8. Ozawa T, Kaihara A, Sato M, Tachihara K, Umezawa Y (2001) Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing. Anal Chem 73:2516–2521PubMedCrossRefGoogle Scholar
  9. 9.
    9. Luker KE, Smith MC, Luker GD, Gammon ST, Piwnica-Worms H, Piwnica-Worms D (2004) Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA 101:12288–12293PubMedCrossRefGoogle Scholar
  10. 10.
    10. Ozawa T, Umezawa Y (2001) Detection of protein-protein interactions in vivo based on protein splicing. Curr Opin Chem Biol 5:578–583PubMedCrossRefGoogle Scholar
  11. 11.
    11. Ozawa T, Nogami S, Sato M, Ohya Y, Umezawa Y (2000) A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. Anal Chem 72:5151–5157PubMedCrossRefGoogle Scholar
  12. 12.
    12. Hu CD, Kerppola TK (2003) Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol 21:539–545PubMedCrossRefGoogle Scholar
  13. 13.
    13. Kaihara A, Kawai Y, Sato M, Ozawa T, Umezawa Y (2003) Locating a protein-protein interaction in living cells via split Renilla luciferase complementation. Anal Chem 75:4176–4181PubMedCrossRefGoogle Scholar
  14. 14.
    14. Kim SB, Ozawa T, Watanabe S, Umezawa Y (2004) High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase. Proc Natl Acad Sci USA 101:11542–11547PubMedCrossRefGoogle Scholar
  15. 15.
    15. Paulmurugan R, Gambhir SS (2005) Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem 77:1295–1302PubMedCrossRefGoogle Scholar
  16. 16.
    16. Paulmurugan R, Gambhir S (2003) Monitoring protein-protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal Chem 75:1584–1589PubMedCrossRefGoogle Scholar
  17. 17.
    17. Wilson T, Hastings JW (1998) Bioluminescence. Annu Rev Cell Dev Biol 14:197–230PubMedCrossRefGoogle Scholar
  18. 18.
    18. Pichler A, Prior J, Piwnica-Worms D (2004) Imaging reversal of multidrug resistance in living mice with bioluminescence: MDR1 P-glycoprotein transports coelenterazine. Proc Natl Acad Sci USA 101:1702–1707PubMedCrossRefGoogle Scholar
  19. 19.
    19. Tarpey M, White C, Suarez E, Richardson G, Radi R, Freeman B (1999) Chemiluminescent detection of oxidants in vascular tissue. Lucigenin but not coelenterazine enhances superoxide formation. Circ Res 84:1203–1211PubMedGoogle Scholar
  20. 20.
    20. Chen J, Zheng X, Brown E, Schreiber S (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA 92:4947–4951PubMedCrossRefGoogle Scholar
  21. 21.
    21. Ostermeier M, Nixon A, Shim J, Benkovic S (1999) Combinatorial protein engineering by incremental truncation. Proc Natl Acad Sci USA 96:3562–3567PubMedCrossRefGoogle Scholar
  22. 22.
    22. Wolff JA, and Budker V (2005) The mechanism of naked DNA uptake and expression. Adv Genet 54:3–20PubMedGoogle Scholar
  23. 23.
    23. Hagstrom JE (2003) Plasmid-based gene delivery to target tissues in vivo: The intravascular approach. Curr Opin Mol Ther 5:338–344PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Victor Villalobos
    • 1
  • Snehal Naik
    • 1
  • David Piwnica-Worms
    • 1
  1. 1.Molecular Imaging Center, Mallinckrodt Institute of Radiology, and Department of Molecular Biology and PharmacologyWashington University School of MedicineSt. LouisUSA

Personalised recommendations