Abstract
A split luciferase complementation assay to study protein–protein interactions within Arabidopsis protoplasts in 96-well plates is described in this protocol. Two proteins of interest, a bait and prey, which are genetically fused to amino- and carboxy-terminal fragments of Renilla luciferase, are transiently expressed in protoplasts. Physical interactions of these bait and prey proteins reconstitute some of the luciferase activity and result in light emission in the presence of the luciferase substrate. This luminescence is then measured by a microplate luminometer. Amounts of the bait protein accumulated in the protoplasts can be estimated by Western blotting using an antibody that recognizes the amino-terminal fragment of Renilla luciferase. The most advantageous aspect of this assay is its capacity of detecting both association and dissociation of a protein pair of interest in a large-scale format.
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References
Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., and Umezawa, Y. (2001) Split luciferase as an optical probe for detecting protein–protein interactions in mammalian cells based on protein splicing. Anal Chem 73, 2516–2521.
Paulmurugan, R., Umezawa, Y., and Gambhir, S. S. (2002) Noninvasive imaging of protein–protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc Natl Acad Sci USA 99, 15608–15613.
Wilson, T. and Hastings, J. W. (1998) Bioluminescence. Annu Rev Cell Dev Biol 14, 197–230.
Fujikawa, Y. and Kato, N. (2007) Split luciferase complementation assay to study protein–protein interactions in Arabidopsis protoplasts. Plant J 52, 185–195.
Stefan, E., Aquin, S., Berger, N., Landry, C. R., Nyfeler, B., Bouvier, M., and Michnick, S. W. (2007) Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo. Proc Natl Acad Sci USA 104, 16916–16921.
Kaihara, A., Kawai, Y., Sato, M., Ozawa, T., and Umezawa, Y. (2003) Locating a protein–protein interaction in living cells via split Renilla luciferase complementation. Anal Chem 75, 4176–4181.
Luker, K. E., Smith, M. C., Luker, G. D., Gammon, S. T., Piwnica-Worms, H., and 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, 12288–12293.
Paulmurugan, R. and Gambhir, S. S. (2003) Monitoring protein-protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal Chem 75, 1584–1589.
Paulmurugan, R., Massoud, T. F., Huang, J., and Gambhir, S. S. (2004) Molecular imaging of drug-modulated protein–protein interactions in living subjects. Cancer Res 64, 2113–2119.
Kim, S. B., Otani, Y., Umezawa, Y., and Tao, H. (2007) Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferase. Anal Chem 79, 4820–4826.
Remy, I. and Michnick, S. W. (2006) A highly sensitive protein–protein interaction assay based on Gaussia luciferase. Nat Methods 3, 977–979.
Chen, H., Zou, Y., Shang, Y., Lin, H., Wang, Y., Cai, R., Tang, X., and Zhou, J. M. (2008) Firefly luciferase complementation imaging assay for protein–protein interactions in plants. Plant Physiol 146, 368–376.
Yoo, S. D., Cho, Y. H., and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat Protoc 2, 1565–1572.
Sambrook, J., and Rusell, D. W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Labortory Press, Cold Spring Harbor, NY.
Acknowledgments
The project was supported by the USDA Cooperative State Research, Education and Extension Service – National Research Initiative – Plant Genome Program, award no. 2006-35604-16627, for N.K. We thank Mr. R. Blake Crochet for his editorial work on the manuscript.
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Kato, N., Jones, J. (2010). The Split Luciferase Complementation Assay. In: Hennig, L., Köhler, C. (eds) Plant Developmental Biology. Methods in Molecular Biology, vol 655. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-765-5_24
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DOI: https://doi.org/10.1007/978-1-60761-765-5_24
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