Abstract
Bioluminescence resonance energy transfer (BRET) is a prominent biophysical technology for monitoring molecular interactions, and has been widely used to study protein-protein interactions (PPI) in live cells. This technology requires proteins of interest to be associated with an energy donor (i.e., luciferase) and an acceptor (e.g., fluorescent protein) molecule. Upon interaction of the proteins of interest, the donor and acceptor will be brought into close proximity and energy transfer of chemical reaction-induced luminescence to its corresponding acceptor will result in an increased emission at an acceptor-defined wavelength, generating the BRET signal. We leverage the advantages of the superior optical properties of the NanoLuc® luciferase (NLuc) as a BRET donor coupled with Venus, a yellow fluorescent protein, as acceptor. We term this NLuc-based BRET platform “BRETn”. BRETn has been demonstrated to have significantly improved assay performance, compared to previous BRET technologies, in terms of sensitivity and scalability. This chapter describes a step-by-step practical protocol for developing a BRETn assay in a multi-well plate format to detect PPIs in live mammalian cells.
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References
Xu Y, Kanauchi A, von Arnim AG, Piston DW, Johnson CH (2003) Bioluminescence resonance energy transfer: monitoring protein-protein interactions in living cells. Methods Enzymol 360:289–301
Pfleger KDG, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3(3):165–174. doi:10.1038/Nmeth841
Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A 96(1):151–156
Couturier C, Deprez B (2012) Setting up a bioluminescence resonance energy transfer high throughput screening assay to search for protein/protein interaction inhibitors in mammalian cells. Front Endocrinol 3:1–13. doi:10.3389/fendo.2012.00100
Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7(11):1848–1857. doi:10.1021/Cb3002478
Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20(1):87–90. doi:10.1038/nbt0102-87
Mo X-L, Luo Y, Ivanov AA, Su R, Havel JJ, Li Z, Khuri F, Du Y, Fu H (2015) Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high throughput biosensor platform. J Mol Cell Biol. doi: 10.1093/jmcb/mjv064
Johnson R, Halder G (2014) The two faces of Hippo: targeting the Hippo pathway for regenerative medicine and cancer treatment. Nat Rev Drug Discov 13(1):63–79. doi:10.1038/nrd4161
Harvey KF, Zhang X, Thomas DM (2013) The Hippo pathway and human cancer. Nat Rev Cancer 13(4):246–257. doi:10.1038/nrc3458
Huang J, Wu S, Barrera J, Matthews K, Pan D (2005) The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP. Cell 122(3):421–434. doi:10.1016/j.cell.2005.06.007
Zhang Z, Lin Z, Zhou Z, Shen HC, Yan SF, Mayweg AV, Xu Z, Qin N, Wong JC, Zhang Z, Rong Y, Fry DC, Hu T (2014) Structure-based design and synthesis of potent cyclic peptides inhibiting the YAP-TEAD protein-protein interaction. ACS Med Chem Lett 5(9):993–998. doi:10.1021/ml500160m
Liu-Chittenden Y, Huang B, Shim JS, Chen Q, Lee SJ, Anders RA, Liu JO, Pan D (2012) Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP. Genes Dev 26(12):1300–1305. doi:10.1101/gad.192856.112
Acknowledgments
We thank Dr. Kun-Liang Guan for providing YAP1, and Dr. Atsushi Miyawaki for providing Venus cDNA plasmid as cloning template. We would like to thank Drs. Jonathan Havel and Zenggang Li for their contributions in generating NLuc destination vector and Venus-YAP1 construct. We also thank Dr. Yuhong Du for her constructive inputs to make this assay work, and Dr. Margaret Johns for editing the text. This study is supported in part by National Cancer Institute to H.F. (NIH U01CA168449) and to the Winship Cancer Institute of Emory University (NIH 5P30CA138292).
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Mo, XL., Fu, H. (2016). BRET: NanoLuc-Based Bioluminescence Resonance Energy Transfer Platform to Monitor Protein-Protein Interactions in Live Cells. In: Janzen, W. (eds) High Throughput Screening. Methods in Molecular Biology, vol 1439. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3673-1_17
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DOI: https://doi.org/10.1007/978-1-4939-3673-1_17
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