BRET: NanoLuc-Based Bioluminescence Resonance Energy Transfer Platform to Monitor Protein-Protein Interactions in Live Cells
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 wordsBioluminescence resonance energy transfer (BRET) Nanoluc luciferase (NLuc) Hippo signaling pathway YAP-TEAD interaction Ultra high-throughput screening (uHTS)
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).
- 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
- 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
- 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