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Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein.The name was first used by Raphaël Dubois who invented the words luciferin and luciferase, for the substrate and enzyme, respectively. [1]
Within the field of molecular biology, a protein-fragment complementation assay, or PCA, is a method for the identification and quantification of protein–protein interactions. In the PCA, the proteins of interest ("bait" and "prey") are each covalently linked to fragments of a third protein (e.g. DHFR, which acts as a "reporter").
Firefly luciferase is the light-emitting enzyme responsible for the bioluminescence of fireflies and click beetles. The enzyme catalyses the oxidation of firefly luciferin , requiring oxygen and ATP .
Luciferase systems are widely used in genetic engineering as reporter genes, each producing a different color by fluorescence, [76] [77] and for biomedical research using bioluminescence imaging. [ 78 ] [ 79 ] [ 80 ] For example, the firefly luciferase gene was used as early as 1986 for research using transgenic tobacco plants. [ 81 ]
It consists of a modified cell line that has been stably transfected with a DNA construct with a luciferase reporter gene under control of receptor-specific DNA response elements that can stimulate transcription of the inserted luciferase gene and produce the light-generating enzyme which can be easily measured. The DNA response elements can be ...
ATP is a molecule found only in and around living cells, and as such it gives a direct measure of biological concentration and health. ATP is quantified by measuring the light produced through its reaction with the naturally-occurring firefly enzyme luciferase using a luminometer.
Common applications include luciferase-based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of ATP. [ 10 ] Time-resolved fluorescence (TRF)
In addition, this study was the first report of an in vivo technique, now known as the bimolecular fluorescence complementation (BiFC) assay, to provide insight into the structural basis of protein complex formation through detection of fluorescence caused by the assembly of fluorescent reporter protein fragments tethered to interacting proteins.