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BRET-linked ATP assay with luciferase
Golnaz Borghei1, Elizabeth A H Hall
1Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK. lisa.hall@biotech.cam.ac.uk.
The Analyst
|May 22, 2014
Summary
Researchers developed a novel bioluminescence resonance energy transfer (BRET) method for sensitive adenosine triphosphate (ATP) detection. This technique utilizes a stable luciferase-mCherry pair, enabling robust ATP quantification across a wide concentration range.
Area of Science:
- Biochemistry
- Molecular Biology
- Biotechnology
Background:
- Bioluminescence Resonance Energy Transfer (BRET) is a powerful technique for studying molecular interactions.
- Developing stable and efficient BRET pairs is crucial for sensitive biological assays.
- Adenosine Triphosphate (ATP) is a key indicator of cellular metabolic activity and energy status.
Purpose of the Study:
- To develop and validate a novel BRET-based assay for sensitive ATP determination.
- To couple a pH- and thermo-stable mutant firefly luciferase with the mCherry fluorescent protein for BRET applications.
- To explore two distinct measurement strategies for ATP quantification using the developed BRET system.
Main Methods:
- Construction of a BRET pair using a mutant firefly luciferase and the red-emitting mCherry protein in fused and unfused formats.
- Characterization of light transfer efficiency and spectral properties of the BRET pair.
- Development of two ATP determination methods: a ratiometric technique and an ATP-dependent wavelength shift assay.
Main Results:
- The BRET pair demonstrated efficient light transfer (>40% red-shifted over 600 nm) with mCherry.
- A ratiometric ATP measurement technique showed calibration slope independence of protein concentration but was limited by BRET efficiency.
- An ATP-dependent wavelength shift assay using mCherry emission above 600 nm provided robust ATP determination (10^-11 to 10^-5 M) independent of protein concentration.
Conclusions:
- A novel and robust BRET-based method for ATP determination has been successfully developed.
- The ATP-dependent wavelength shift assay offers a promising approach for sensitive and reliable ATP quantification.
- Further improvements in acceptor quantum yield could enhance the sensitivity of BRET-based ATP detection.
