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.
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References
Michnick, S.W. Curr. Opin. Struct. Biol. 11, 472–477 (2001).
Ghosh, I., Hamilton, A.D. & Regan, L. J. Am. Chem. Soc. 122, 5658–5659 (2000).
Hu, C.D., Chinenov, Y. & Kerppola, T.K. Mol. Cell 9, 789–798 (2002).
Remy, I. & Michnick, S.W. Methods 32, 381–388 (2004).
Remy, I., Montmarquette, A. & Michnick, S.W. Nat. Cell Biol. 6, 358–365 (2004).
Magliery, T.J. et al. J. Am. Chem. Soc. 127, 146–157 (2005).
Macdonald, M.L. et al. Nat. Chem. Biol. 2, 329–337 (2006).
Zhang, J., Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).
Tannous, B.A., Kim, D.E., Fernandez, J.L., Weissleder, R. & Breakefield, X.O. Mol. Ther. 11, 435–443 (2005).
Rost, B. Methods Enzymol. 266, 525–539 (1996).
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).
Paulmurugan, R. & Gambhir, S.S. Anal. Chem. 75, 1584–1589 (2003).
Chen, J., Zheng, X.F., Brown, E.J. & Schreiber, S.L. Proc. Natl. Acad. Sci. USA 92, 4947–4951 (1995).
Banaszynski, L.A., Liu, C.W. & Wandless, T.J. J. Am. Chem. Soc. 127, 4715–4721 (2005).
Rollins, C.T. et al. Proc. Natl. Acad. Sci. USA 97, 7096–7101 (2000).
Conery, A.R. et al. Nat. Cell Biol. 6, 366–372 (2004).
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).
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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)
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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
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DOI: https://doi.org/10.1038/nmeth979
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