Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.
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August 2001
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Research Article|
July 25 2001
Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin Available to Purchase
Jonathan P. WAUD;
Jonathan P. WAUD
1
∗Department of Medical Biochemistry, Cardiff and Vale NHS Trust, Llandough Hospital, Llandough, Penarth, Vale of Glamorgan CF64 2XX, U.K.
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Alexandra BERMÚDEZ FAJARDO;
Alexandra BERMÚDEZ FAJARDO
1
†Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, South Glamorgan CF14 4XN, U.K.
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Thankiah SUDHAHARAN;
Thankiah SUDHAHARAN
†Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, South Glamorgan CF14 4XN, U.K.
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Andrew R. TRIMBY;
Andrew R. TRIMBY
†Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, South Glamorgan CF14 4XN, U.K.
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Jinny JEFFERY;
Jinny JEFFERY
2
†Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, South Glamorgan CF14 4XN, U.K.
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Ann JONES;
Ann JONES
‡Nycomed Amersham plc, Forest Farm Industrial Estate, Cardiff, Vale of Glamorgan CF14 7YT, U.K.
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Anthony K. CAMPBELL
Anthony K. CAMPBELL
3
‡Nycomed Amersham plc, Forest Farm Industrial Estate, Cardiff, Vale of Glamorgan CF14 7YT, U.K.
3To whom correspondence should be addressed (e-mail [email protected]).
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Publisher: Portland Press Ltd
Received:
February 20 2001
Revision Received:
April 03 2001
Accepted:
April 16 2001
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2001
2001
Biochem J (2001) 357 (3): 687–697.
Article history
Received:
February 20 2001
Revision Received:
April 03 2001
Accepted:
April 16 2001
Citation
Jonathan P. WAUD, Alexandra BERMÚDEZ FAJARDO, Thankiah SUDHAHARAN, Andrew R. TRIMBY, Jinny JEFFERY, Ann JONES, Anthony K. CAMPBELL; Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin. Biochem J 1 August 2001; 357 (3): 687–697. doi: https://doi.org/10.1042/bj3570687
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