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.
Abbreviations used: CCD, charge-coupled device; CRET, chemiluminescence resonance energy transfer; EGFP, enhanced green fluorescent protein (GFP human codon usage, with the mutation S65T); EGCAq, EGFP–caspase-3 linker–aequorin; EGTAq, EGFP–α-thrombin linker–aequorin; FMK, fluoromethylketone; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GAq, GFP–aequorin; GCAq, GFP–caspase-3 linker–aequorin; GTAq, GFP–α-thrombin linker–aequorin; LAq, firefly-luciferase–aequorin; Z-, benzyloxycarbonyl.
These authors contributed equally to this paper.
Present address: Combined Laboratory, Plymouth Hospitals NHS Trust, Derriford Hospital, Derriford Road, Plymouth PL6 8DH, U.K.