NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells

Dixon, Andrew S.; Schwinn, Marie K.; Hall, Mary P.; Zimmerman, Kris; Otto, Paul; Lübben, Thomas; Butler, Braeden L.; Binkowski, Brock F.; Machleidt, Thomas; Kirkland, Thomas A.; Wood, Monika G.; Eggers, Christopher T.; Encell, Lance P.; Wood, Keith V.

doi:10.1021/acschembio.5b00753

Cited by 1,118 publications

(1,344 citation statements)

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“…In G-protein coupled receptor (GPCR) drug screening, receptor-G protein coupling as well as G protein subunit rearrangement can be used effectively as ways to measure agonist and antagonist behaviors [12]. While this manuscript was in preparation, a thorough analysis of split positions for NanoLuc was reported [13]. The split position we selected in this current work happens to be near the residues tested by Dixon etal .…”

Section: Introductionmentioning

confidence: 99%

Luciferase Complementation Based-Detection of G-Protein-Coupled Receptor Activity

et al. 2018

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Protein complementation assays (PCA) are used as pharmacological tools, enabling a wide array of applications, ranging from studies of protein-protein interactions to second messenger effects. Methods to detect activities of G protein-coupled receptors (GPCRs) have particular relevance for drug screening. Recent development of an engineered luciferase NanoLuc created the possibility of generating a novel PCA, which in turn could open a new avenue for developing drug screening assays. Here we identified a novel split position for NanoLuc and demonstrated its use in a series of fusion constructs to detect the activity of GPCRs. The split construct can be applied to a variety of pharmacological screening systems.

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Section: Introductionmentioning

confidence: 99%

Luciferase Complementation Based-Detection of G-Protein-Coupled Receptor Activity

et al. 2018

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“…For example, although not yet tested in plants, the design of the NanoBiT system has involved determining the K d of the free fragments, characterizing their turnover rates, and documenting differences in steric accessibility (Dixon et al, 2016).…”

Section: Resultsmentioning

confidence: 99%

“…Table IV provides an overview of the different systems used in planta, where the luciferases from the North American firefly (Photinus pyralis) and the sea pansy (Renilla reniformis) are utilized exclusively, while in mammalian cells, other luciferases derived from click beetle, Gaussia spp. and nanoLUC also have been used (Remy and Michnick, 2006;Kim et al, 2007;Dixon et al, 2016).…”

Section: Split-luciferase Complementation Assay: Let There Be Lightmentioning

confidence: 99%

“…Implementation of other luciferases (Gaussia spp., click beetle, or nanoLUC), which may be more suitable for the demands of plant systems, will likely pull the splitluciferase complementation assay out of its niche existence. In fact, the nanoLuc split-luciferase complementation system NanoBiT, which has been engineered to be ideal for most PPIs (Dixon et al, 2016), will hopefully be available for plant scientists in the near future.…”

Section: Firefly Versus Renilla: Comparison Of Propertiesmentioning

confidence: 99%

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Techniques for the analysis of protein-protein interactions in vivo

et al. 2016

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ORCID ID: 0000-0002-5820-4466 (C.G.).Identifying key players and their interactions is fundamental for understanding biochemical mechanisms at the molecular level. The ever-increasing number of alternative ways to detect protein-protein interactions (PPIs) speaks volumes about the creativity of scientists in hunting for the optimal technique. PPIs derived from single experiments or high-throughput screens enable the decoding of binary interactions, the building of large-scale interaction maps of single organisms, and the establishment of crossspecies networks. This review provides a historical view of the development of PPI technology over the past three decades, particularly focusing on in vivo PPI techniques that are inexpensive to perform and/or easy to implement in a state-of-the-art molecular biology laboratory. Special emphasis is given to their feasibility and application for plant biology as well as recent improvements or additions to these established techniques. The biology behind each method and its advantages and disadvantages are discussed in detail, as are the design, execution, and evaluation of PPI analysis. We also aim to raise awareness about the technological considerations and the inherent flaws of these methods, which may have an impact on the biological interpretation of PPIs. Ultimately, we hope this review serves as a useful reference when choosing the most suitable PPI technique.

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“…Next, important design considerations will be discussed in more detail by describing the development of different classes of BRET-sensors, both from our own work and that of others. These examples are all based on the NanoLuc luciferase, a bright and very stable blue light-emitting luciferase developed by Promega that has quickly risen to prominence in the bioluminescence field (Dixon et al, 2016;England, Ehlerding, & Cai, 2016;Hall et al, 2012;Machleidt et al, 2015;Stoddart et al, 2015).…”

Section: Introductionmentioning

confidence: 99%