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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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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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)
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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
