Genetic Engineering & Biotechnology News

MAY1 2015

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Genetic Engineering & Biotechnology News | | MAY 1, 2015 | 17 relative expression of the fusion donor protein to the fusion protein acceptor is important for obtaining the optimal NanoBRET signal, as the amount of unbound donor within the cell should be minimized. NanoBRET protein:protein interactions can then be measured from live cells in either 96- or 384-well format using an instrument capable of measuring dual fltered signals. NanoBRET, like other BRET assays, is a ra- tiometric assay with the calculation shown in Figure 1B. Inherent to ratiometric assays and observed with NanoBRET assay are sev- eral advantages for screening; a high level of robustness results in high Z factors and low CVs, an independence of cell number, and in- sulation of data variance due to slight changes in assay setup or plating. The NanoBRET assay can be used to understand how a specifc protein:protein in- teraction is modulated upon treatment of the cells with different stimuli (Figure 2). We used full-length proteins of human p53 fusion as donor and human MDM2 fusion as accep- tor, expressed in HEK293 cells to show the ability to measure the interaction and specifc inhibition of the interaction with Nutlin 3A (Figure 2A). These live-cell measurements are used to determine the IC50 and they showed high Z factor values, which are important for screening. One can also perform inhibi- tion studies by using the defned N-terminal region of p53 (amino acids 1-118), which is known to mediate the interaction with MDM2 (Figure 2B). A similar in-cell IC50 value is obtained when using the p53 N-terminal domain in the NanoBRET assay, compared to full-length proteins, indicating that this terminal region is indeed important for the interaction and re- sponse to inhibitor (Figures 2A and 2B). Since NanoBRET method works in live cells, kinetic and real-time measurements can be performed to understand the dynamic na- ture of protein interactions. NanoBRET assay optimization of the membrane protein recep- tor, EGFR, with its adaptor protein Grb2 is shown in Figure 2C. As expected, this interac- tion is increased upon addition of the growth ligand for EGFR, EGF, with a known EC50 value. Figure 2D demonstrates the ability to monitor the rate of increase of this interaction immediately after addition of EGF, which oc- curs within seconds inside the cell. We have developed and optimized over 100 protein:protein interaction NanoBRET assays, which are available from Promega. Table 1 shows a highlighted list of currently available NanoBRET assays, many of which cover important areas of drug targeting in epigenetics, transcription, and receptor biol- ogy. A signifcant number of pre-built Nano- BRET assays are for monitoring interactions of bromodomain proteins with chromatin, many of which have been qualifed by exist- ing inhibitors. As most inhibitors to these targets are initially found using the bromodomain frag- ment alone, several of the assays include bro- modomains along with the full-length pro- tein as indicated. For many of these family members, we have observed inhibition from chromatin of the bromodomain alone can be achieved inside the cell, mimicking the in vitro screening conditions, but this does not always translate to inhibition or eviction of the full-length protein by the inhibitor. This is likely due to the complexity of interactions and the multiple points of contact with chro- matin that are present within the full-length proteins, which these cellular NanoBRET as- says can begin to deconvolute. VIAFLO ASSIST Convert your multichannel pipette into an automated system for best results and unmatched ergonomics. READY. .. SET. .. P IPETTE! click DRUG DISCOVERY Assay Tutorial These predesigned, full-length protein NanoBRET assays are available for expression in mammalian cells. Bromodomain assays, which are available as bromodomain-alone vectors, are indicated with an asterisk (*). Table 1. Available Predesigned Assays Bromodomain Targets Other Epigenetic Targets Transcriptional Targets BRD2/Histone H3.3 or H4 EZH2/Histone H3.1 or H3.3 P300/HIF1alpha BRD3/Histone H3.3 or H4 EZH2/SUZ12 P53/MDM2 BRD4*/Histone H3.3 or H4 EZH2/EED1 N-terminal p53/MDM2 CBP*/ Histone H3.3 or H4 EED1/Histone H3.1 or H3.3 P53/PARP1 ATAD2A*/Histone H3.3 or H4 SETD1A/Histone H3.3 MED12/MED26 BRDT/Histone H3.3 or H4 HDAC1/HDAC2 MED13/MED26 BRPF1*/Histone H3.3 or H4 SETD1A/Histone H3.3 cMYC/MAX BRPF2/Histone H3.3 or H4 L3MBTL3 3MBT/Histone H4 Signaling Pathways and Kinases BRPF3/Histone H3.3 or H4 MLL1/Menin FKBP/FRB BRD7*/Histone H3.3 or H4 TET3/Histone H2A, H2B, H3.3 or H4 KRas(G12C)/bRaf CECR2*/Histone H3.3 or H4 LSH/Histone H2A, H2B, H3.3 or H4 KRas/bRaf GCN5/Histone H3.3 or H4 OGT/Histone H2A or H2B KRas/cRaf PCAF*/Histone H3.3 or H4 CBX1/Histone H3.1 MEK/ERK EP300*/Histone H3.3 or H4 CBX2/Histone H3.1 SKP1/SKP2 MLL1/Histone H3.3 or H4 CBX3/Histone H3.1 SKP2/p27 SMARCA2/Histone H3.3 or H4 CBX5/Histone H3.1 SKP2/CKS1B BRD9*/Histone H3.3 or H4 LSD1/H3.3 Membrane Proteins BAZ2A*/Histone H3.3 or H4 KDM4C TUDOR/H3.3 EGFR/GRB2 SMARCA4/Histone H3.3 or H4 BRD4/B-catenin MRAP/MC2R TAF1*/Histone H3.3 BRD4/CDK9 MRAP/MRAP Danette L. Daniels, Ph.D. (danette., is a group leader, Thomas Machleidt, Ph.D., is head of cell biology in advanced technologies, Jacqui Méndez is a senior research scientist, Kristin Riching, Ph.D., is a postdoctoral fellow, Marie Schwinn, Ph.D., is a senior research scientist, Nancy Murphy is a research sci- entist, Thomas Kirkland, Ph.D., is head of advanced technology chemistry research, Keith Wood, Ph.D., is head of research, and Marjeta Urh, Ph.D., is director of R&D research at Promega, which supplies assay and reagent materials as well as custom as- say services. Website: Figure 2. NanoBRET assays showing inhibition, activation, and real-time measurements in living cells. (A) NanoBRET assay of full-length p53 and MDM2 proteins shows inhibition with Nutlin-3A. (B) NanoBRET assay of an N-terminal fragment (amino acids 1-118) and full-length MDM2 showing inhibition with Nutlin-3A. (C) NanoBRET assay of full-length EGFR and Grb-2 showing induction of interaction using the growth factor, EGF. (D) Real-time NanoBRET assay of EGFR and Grb2 showing kinetic induction of interaction upon addition of EGF. All graphs show calculated BRET ratio units*1000 (mBU) on the y-axis and indicated drug treatment on the x-axis (A, B, and C) or the time of treatment (D). A B C D

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