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BRET: NanoLuc-Based Bioluminescence Resonance Energy Transfer Platform to Monitor Protein-Protein Interactions in Live Cells

  • Xiu-Lei MoEmail author
  • Haian Fu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1439)

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.

Key words

Bioluminescence resonance energy transfer (BRET) Nanoluc luciferase (NLuc) Hippo signaling pathway YAP-TEAD interaction Ultra high-throughput screening (uHTS) 

Notes

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).

References

  1. 1.
    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 360:289–301CrossRefGoogle Scholar
  2. 2.
    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 CrossRefGoogle Scholar
  3. 3.
    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):151–156CrossRefGoogle Scholar
  4. 4.
    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 CrossRefGoogle Scholar
  5. 5.
    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 CrossRefGoogle Scholar
  6. 6.
    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 CrossRefGoogle Scholar
  7. 7.
    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/mjv064Google Scholar
  8. 8.
    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 CrossRefGoogle Scholar
  9. 9.
    Harvey KF, Zhang X, Thomas DM (2013) The Hippo pathway and human cancer. Nat Rev Cancer 13(4):246–257. doi: 10.1038/nrc3458 CrossRefGoogle Scholar
  10. 10.
    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 CrossRefGoogle Scholar
  11. 11.
    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 CrossRefGoogle Scholar
  12. 12.
    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 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Pharmacology and Emory Chemical Biology Discovery CenterEmory University School of MedicineAtlantaUSA
  2. 2.Department of Hematology and Medical Oncology and Winship Cancer InstituteEmory UniversityAtlantaUSA

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