ArticleThe Biochemical journal2001
Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin.
Article in The Biochemical journal, 2001. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
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Who cites it
8 citing papers in PubMed.
- Small Molecule Active Site Directed Tools for Studying Human Caspases.Chemical reviews · 2015Review
- A novel luminescence-based method for the detection of functionally active antibodies to muscarinic acetylcholine receptors of the M3 type (mAchR3) in patients' sera.Clinical and experimental immunology · 2014Article
- An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.Open biology · 2014Article
- Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.PloS one · 2011Article
- Biosensing and imaging based on bioluminescence resonance energy transfer.Current opinion in biotechnology · 2009Review
- The molecular basis of lactose intolerance.Science progress · 2009Review
- Microscopy and image analysis.Current protocols in human genetics · 2005Article
- The molecular basis of lactose intolerance.Science progress · 2005Review
Corrections and comments
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Authors and funding
7 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 (alpha-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 2 microM). 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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