Engineering with NanoLuc: a playground for the development of bioluminescent protein switches and sensors

2020 ◽  
Vol 48 (6) ◽  
pp. 2643-2655
Author(s):  
Lieuwe Biewenga ◽  
Bas J.H.M. Rosier ◽  
Maarten Merkx
Keyword(s):  
Energy Transfer ◽  
Cell Biology ◽  
Chemical Biology ◽  
Dynamic Range ◽  
State Of The Art ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Advantages And Disadvantages ◽  
Bioluminescent Protein

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

scholarly journals An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)

2019 ◽  
Author(s):  
Eric J. Aird ◽  
Kassidy J. Tompkins ◽  
Wendy R. Gordon
Keyword(s):  
Energy Transfer ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cellular Protein ◽  
Bioluminescence Resonance Energy Transfer ◽  
Tension Sensor ◽  
Focal Adhesion Protein

ABSTRACTMolecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

Antibody engineering-driven controllable chemiluminescence resonance energy transfer for immunoassay with tunable dynamic range

2021 ◽  
Vol 1152 ◽  
pp. 338231
Author(s):  
Leina Dou ◽  
Yantong Pan ◽  
Mingfang Ma ◽  
Suxia Zhang ◽  
Jianzhong Shen ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antibody Engineering

scholarly journals Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

scholarly journals Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET)

2011 ◽  
Vol 29 (1) ◽  
pp. 119-124 ◽  
Author(s):  
Helen Dacres ◽  
Jian Wang ◽  
Virginia Leitch ◽  
Irene Horne ◽  
Alisha R. Anderson ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer
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