Genetic Engineering & Biotechnology News

MAY1 2015

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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16 | MAY 1, 2015 | GENengnews.com | Genetic Engineering & Biotechnology News Danette L. Daniels, Ph.D., Thomas Machleidt, Ph.D., Jacqui Méndez, Kristin Riching, Ph.D., Marie Schwinn, Ph.D., Nancy Murphy, Thomas Kirkland, Ph.D., Keith Wood, Ph.D., and Marjeta Urh, Ph.D. Signifcant advances in the understanding of the human proteomics network have ad- vanced knowledge of the roles of specifc protein:protein interactions, particularly re- lated to disease and disease progression. This has resulted in an increased effort to target particular protein:protein interactions or monitor protein:protein nodules within sig- nalling pathways in numerous areas of drug discovery research. To successfully do so, technology with a high degree of specifcity and sensitivity is needed to detect protein interactions and their dynamic changes within cells. One such technology capable of this is bioluminescence resonance energy transfer, BRET, which mea- sures energy transfer from a luminescent do- nor to a fuorescent acceptor within a defned distance range between appropriately labelled proteins (Figure 1A). Current BRET assay systems however lack the necessary sensitivity and are not able to de- tect subtle changes of protein:protein interac- tions. The main issues are the lack of donor brightness and the close proximity of donor and acceptor emission spectra that result in poor sensitivity and limited dynamic range. Compounding the issue is the need to express the donor luciferase at high levels to achieve effcient energy transfer to the acceptor, which can in certain cases lead to altered biological response. Here we present a new confgura- tion of BRET, termed NanoBRET™, which addresses these challenges through the use of a NanoLuc ® luciferase donor in combination with a fuorescently labelled HaloTag ® accep- tor (Figures 1A and 1B). The NanoBRET combination of a bright donor with a spectrally well-separated accep- tor results in reduced donor background in the acceptor channel and leads to signifcant im- provements in sensitivity and dynamic range (Figure 1B). NanoLuc luciferase is >100-fold brighter than other luciferases, and therefore expression of the NanoLuc donor protein in the assay can be lowered to levels of the endogenous protein expression. Appropriate expression of the protein (i.e., not highly over- expressed) is important for obtaining a proper physiological response and for improving the overall signal:background compared to other BRET donor:acceptor confgurations. In the NanoBRET method, the two pro- teins of interest are expressed in a mamma- lian cell line of choice as either NanoLuc or HaloTag protein fusions. It is important that fusions are created in optimal physiological orientation and spatial proximity in order to achieve optimal energy transfer. This is typi- cally determined experimentally. Similarly, the Using NanoBRET Technology to Follow Changes in Intracellular Protein Binding Events Illuminating Protein:Protein Interactions in Living Cells DRUG DISCOVERY Assay Tutorial Figure 1. Principle of the NanoBRET assay for detecting intracellular protein:protein interactions. (A) The NanoBRET assay is a proximity based assay measuring energy transfer between a NanoLuc donor fusion protein and a fuorescently labelled HaloTag acceptor fusion protein. (B) Spec tral separation of donor and acceptor signals in the NanoBRET assay leads to reduced background within the assay and calculation of the NanoBRET ratio, which is the acceptor signal divided by the donor signal. Indicated are the emission wavelength of the NanoLuc (peak at 450 nM) and the fluorescent HaloTag 618 ligand (peak at 618 nM), as well as the region wherein BRET is measured. A B

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