Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays

Auld, Douglas S.; Narahari, Janaki; Ho, Pei-i; Casalena, Dominick; Nguyen, Vy; Cirbaite, Evelina; Hughes, Douglas E.; Daly, John D.; Webb, Brian

doi:10.1021/acs.biochem.8b00290

Cited by 19 publications

(22 citation statements)

References 26 publications

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“…2) with a molecular mass of 9-10 kDa corresponding to GpLuc tandem repeats was identified. Complementation of the N-and C-terminal fragments of GpLuc resulted in reconstitution of only 10% of the initial activity, but this value was comparable to or exceeded the specific activity of intact RLuc and FLuc (21,55). In contrast to the results of the study (27), the fragments separately did not exhibit any bioluminescent activity and revealed reversible complementation, enabling quantitative measurement of dynamic and transient protein-protein interactions.…”

Section: Protein-fragment Complementation Assaymentioning

confidence: 72%

“…), was constructed on the base of conservative catalytic parts of MpLuc1 from Metridia pacifica and MLuc164 from M. longa . In terms of activity, TurboLuc was comparable with wt Gaussia luciferase but had a 10‐nm red shift as compared to 470‐nm peak of wt GpLuc at the same assay conditions .…”

Section: Improvement Of Reporter Properties By Mutagenesismentioning

confidence: 88%

“…This obstacle can be overcome by optimizing buffer conditions and obtaining the improved luciferases with glow‐type luminescence by mutagenesis. The mutant TurboLuc reported with slow luminescent signal decay was specifically designed for HTS on the base of Metridia luciferase family and successfully applied for screening of low molecular weight compound libraries . The GpLuc glow mutant M60L/M127L fused with secreted protein extracellular matrix fibulin‐3 was successfully used for HTS identification of small molecules, modulators of fibulin‐3 secretion the loss of which in retinal cell lines is associated with macular degeneration .…”

Section: Application Of Copepod Luciferasesmentioning

confidence: 99%

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Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Markova

Larionova

Vysotski

2019

Photochem & Photobiology

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Copepod luciferases—a family of small secretory proteins of 18.4–24.3 kDa, including a signal peptide—are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine‐dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high‐throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

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Section: Protein-fragment Complementation Assaymentioning

confidence: 72%

Section: Improvement Of Reporter Properties By Mutagenesismentioning

confidence: 88%

Section: Application Of Copepod Luciferasesmentioning

confidence: 99%

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Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Markova

Larionova

Vysotski

2019

Photochem & Photobiology

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“…Some compounds increase enzymatic activity, while others decrease it. The TurboLuc system reported by Audi et al is somewhat smaller in molecular weight (16 kDa) than NanoLuc [73]. Compared with NanoLuc Protein-Protein Interaction Assays Using Split-NanoLuc DOI: http://dx.doi.org /10.5772/intechopen.86122 and Firefly luciferase, the activity of TurboLuc was less affected by low-molecular weight compounds.…”

Section: Discussionmentioning

confidence: 98%

Protein-Protein Interaction Assays Using Split-NanoLuc

Ohmuro‐Matsuyama¹,

Ueda²

2019

Bioluminescence - Analytical Applications and Basic Biology

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Protein-protein interaction assays are fundamental to basic biology, drug discovery, diagnostics, screening, and immunoassays. Protein-fragment complementation (PCA) is one of such useful protein-protein interaction assays. PCA when performed using luciferase is a reversible approach, whereas when performed using green fluorescent protein analogs is an irreversible approach. The NanoLuc technology developed in 2012 utilizes a small and structurally robust luciferase that is capable of producing very bright luminescence. NanoLuc PCA has been used to detect many protein-protein interactions and for screening purposes. Methods developed from NanoLuc PCA include the HiBiT technology and NanoLuc ternary technology. These novel technologies are promising in various fields and further developments are anticipated.Bioluminescence -Analytical Applications and Basic Biology 2 very bright luminescence. Based on this attractive enzyme, PCA systems were developed [13,14]. This innovation on the NanoLuc PCA improves the luminescent signal, which is markedly better than the conventional PCA signal obtained using other luciferases. Herein, we will focus on the new PCA technology and its application, and further discuss potential improvements in the system. PCA using NanoLucVerhoef et al. constructed a PCA system using NanoLuc [14]. They made several pairs of NanoLuc fragments by cutting at several loop regions, and selected a pair comprised of the N-terminal 52-amino acid (aa) fragment and the C-terminal 119-aa fragment (Figure 2A). These fragments were used to successfully detect the interaction between the transactivation domain fragment of p53 and Mdm2.At almost the same time, Dixon et al. developed another NanoLuc-based PCA system designated NanoLuc Binary Technology (NanoBiT) [13]. This was devised by first identifying a dissection site from 90 candidate sites. An 18-kDa N-terminal fragment and 13-aa C-terminal fragment were selected. The K D value between these fragments was 6 μM. This low affinity was suitable for PCA, but their use was hampered by the very low stability of the N-terminal fragment. The sequence of the N-terminal fragment was optimized from an N-terminal library containing 15,000 variants. The optimization increased the luminescent signal by 300-fold when the two fragments were interacting, which was 37% that of the wild-type NanoLuc. However, the affinity between the N-and C-terminal fragments became too strong for PCA (K D = 900 nM). As a next step, the sequence of the C-terminal peptide was optimized from 350 variants. Finally, two fragments were obtained. They were designated LgBiT (18 kDa) and SmBiT (11 aa). These exhibited significantly low

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“…uHTS in 1536-well microtiter plate (1536w-MTP) formats can enable assay volumes of less than 5 μL and a throughput of more than a 100,000 assays per day [ 36 ]. Auld et al [ 37 ] screened two low molecular weight compound libraries (44,000 compounds) and a purified natural products library (2583 compounds) to describe the inhibitor profile of purified enzyme, TurboLuc, and also showed the use of this reporter enzyme in cell-based assays employing uHTS in 1536w-MTP. Norcliffe et al [ 38 ] adopted an auxotrophic yeast-based uHTS approach for inhibitors of the Leishmania enzyme (inositol phosphorylceramide synthase).…”

Section: Well Plate Setupsmentioning

confidence: 99%

Microfluidic Devices for Drug Assays

2018

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In this review, we give an overview of the current state of microfluidic-based high-throughput drug assays. In this highly interdisciplinary research field, various approaches have been applied to high-throughput drug screening, including microtiter plate, droplets microfluidics as well as continuous flow, diffusion and concentration gradients-based microfluidic drug assays. Therefore, we reviewed over 100 recent publications in the field and sorted them according to their microfluidic approach. As a result, we are showcasing, comparing and discussing broadly applied approaches as well as singular promising ones that might contribute to shaping the future of this field.

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Characterization and Use of TurboLuc Luciferase as a Reporter for High-Throughput Assays

Cited by 19 publications

References 26 publications

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

Protein-Protein Interaction Assays Using Split-NanoLuc

Microfluidic Devices for Drug Assays

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