Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A highly sensitive protein-protein interaction assay based on Gaussia luciferase

Abstract

Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chemical reversal, and induction and inhibition of a key interaction linking insulin and TGFβ signaling.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Characterization of the hGLuc PCA.
Figure 2: The hGLuc PCA is inducible and fully reversible.
Figure 3: Endogenous levels of interacting proteins can be detected with the hGLuc PCA.

References

  1. Michnick, S.W. Curr. Opin. Struct. Biol. 11, 472–477 (2001).

    Article  CAS  Google Scholar 

  2. Ghosh, I., Hamilton, A.D. & Regan, L. J. Am. Chem. Soc. 122, 5658–5659 (2000).

    Article  CAS  Google Scholar 

  3. Hu, C.D., Chinenov, Y. & Kerppola, T.K. Mol. Cell 9, 789–798 (2002).

    Article  CAS  Google Scholar 

  4. Remy, I. & Michnick, S.W. Methods 32, 381–388 (2004).

    Article  CAS  Google Scholar 

  5. Remy, I., Montmarquette, A. & Michnick, S.W. Nat. Cell Biol. 6, 358–365 (2004).

    Article  CAS  Google Scholar 

  6. Magliery, T.J. et al. J. Am. Chem. Soc. 127, 146–157 (2005).

    Article  CAS  Google Scholar 

  7. Macdonald, M.L. et al. Nat. Chem. Biol. 2, 329–337 (2006).

    Article  CAS  Google Scholar 

  8. Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  Google Scholar 

  9. Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R. & Breakefield, X.O. Mol. Ther. 11, 435–443 (2005).

    Article  CAS  Google Scholar 

  10. Rost, B. Methods Enzymol. 266, 525–539 (1996).

    Article  CAS  Google Scholar 

  11. Remy, I., Campbell-Valois, F.X., Ghaddar, G., Aquin, S. & Michnick, S.W. In Protein-protein interactions: a molecular cloning manual (2nd edn.) 637–672 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2005).

    Google Scholar 

  12. Paulmurugan, R. & Gambhir, S.S. Anal. Chem. 75, 1584–1589 (2003).

    Article  CAS  Google Scholar 

  13. Chen, J., Zheng, X.F., Brown, E.J. & Schreiber, S.L. Proc. Natl. Acad. Sci. USA 92, 4947–4951 (1995).

    Article  CAS  Google Scholar 

  14. Banaszynski, L.A., Liu, C.W. & Wandless, T.J. J. Am. Chem. Soc. 127, 4715–4721 (2005).

    Article  CAS  Google Scholar 

  15. Rollins, C.T. et al. Proc. Natl. Acad. Sci. USA 97, 7096–7101 (2000).

    Article  CAS  Google Scholar 

  16. Conery, A.R. et al. Nat. Cell Biol. 6, 366–372 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge V. Rivera and T. Clackson (ARIAD Pharmaceuticals, Inc.) for providing AP21998 and FM cDNA. This work was supported by the Canadian Institute of Health Research (MOP-152556).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Stephen W Michnick.

Ethics declarations

Competing interests

S.W.M. is a common stock holder in Odyssey Thera Inc.

Supplementary information

Supplementary Fig. 1

Development of the Gaussia luciferase PCA. (PDF 283 kb)

Supplementary Fig. 2

Reassociation of FM-hGLuc(1)/FM-hGLuc(2) complexes after AP21998 removal. (PDF 112 kb)

Supplementary Fig. 3

Disruption of FM-hGLuc(1)/FM-hGLuc(2) complexes by FK506. (PDF 135 kb)

Supplementary Fig. 4

hGLuc and hRLuc PCAs fold within 60 seconds. (PDF 162 kb)

Supplementary Methods (PDF 87 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Remy, I., Michnick, S. A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3, 977–979 (2006). https://doi.org/10.1038/nmeth979

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth979

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing