Luciferase, firefly

Firefly luciferase inhibition

Firefly luciferase (Luc) is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. Luc catalyzes the bioluminescent reaction involving firefly luciferin (D-LH(2)), adenosine triphosphate (ATP), magnesium ion and molecular oxygen with the formation of an electronically excited species (oxyluciferin), inorganic pyrophosphate (PPi), carbon dioxide and adenosine monophosphate (AMP). Luc also catalyzes other non-luminescent reactions, which can interfere with the light production mechanism. Following electronic relaxation, the excited oxyluciferin emits radiation in the visible region of the electromagnetic spectrum (550-570 nm). Among the various possible compounds, several classes of inhibitory substances interfere with the activity of this enzyme: here, we consider substrate-related compounds, intermediates or products of the Luc catalyzed reactions, in addition to anesthetics and, fatty acids. This review summarizes the main inhibitors of Luc and the corresponding inhibition kinetic parameters.

Firefly Luciferase-based Fusion Proteins and their Applications in Bioanalysis

Firefly luciferase is widely used in molecular biology and bioanalytical systems as a reporter molecule due to the high quantum yield of the bioluminescence, availability of stable mutant forms of the enzyme with prescribed spectral characteristics and abundance of bacterial expression systems suitable for production of recombinant proteins in limitless quantities. In this review, we described fusion proteins of luciferase with biotin-binding domain and streptavidin, with proteins A and G, antibodies, with DNA- and RNA-binding proteins, as well as fusion proteins designed for BRET systems. The firefly luciferase-based fusion proteins are represented as an effective tool for the development of different bioanalytical systems such as (1) systems in which luciferase is attached to the surface of the target and the bioluminescence signal is detected from the specific complexes formed; (2) BRET-based systems, in which the specific interaction induces changes in the bioluminescence spectrum; and (3) systems that use modified or split luciferases, in which the luciferase activity changes under the action of the analyte. All these systems have wide application in biochemical analysis of physiologically important compounds, for the detection of pathogenic bacteria and viruses, for evaluation of protein-protein interactions, assaying of metabolites involved in cell communication and cell signaling.

Promoter deletion analysis using a dual-luciferase reporter system

Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter.Luciferase genes have been widely used as reporter genes for their sensitivity and efficiency. Firefly and Renilla luciferases are two commonly used reporters, which oxidize different substrates to generate quantifiable luminescence. Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions

Firefly luciferase is a member of the acyl-adenylate/thioester-forming superfamily of enzymes and catalyzes the oxidation of firefly luciferin with molecular oxygen to emit light. Knowledge of the luminescence mechanism catalyzed by firefly luciferase has been gathered, leading to the discovery of a novel catalytic function of luciferase. Recently, we demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis from fatty acids in the presence of ATP, Mg(2+) and coenzyme A. Based on identification of fatty acyl-CoA genes in firefly, Drosophila, and non-luminous click beetles, we then proposed that the evolutionary origin of firefly luciferase is a fatty acyl-CoA synthetase in insects. Further, we succeeded in converting the fatty acyl-CoA synthetase of non-luminous insects into functional luciferase showing luminescence activity by site-directed mutagenesis.

Luciferase assay to study the activity of a cloned promoter DNA fragment

Luciferase based assays have become an invaluable tool for the analysis of cloned promoter DNA fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. Here, we describe the use of the Dual-Luciferase(?) Reporter Assay System created by Promega (Promega Corporation, Wisconsin, USA) to study the cloned 6.7 kilobases (kb) mouse (m) Tcf3 promoter DNA fragment in mouse embryonic derived neural stem cells (NSC). In this system, the expression of the firefly luciferase driven by the cloned mTcf3 promoter DNA fragment (including transcription initiation sites) is correlated with a co-transfected control reporter expressing Renilla luciferase from the herpes simplex virus (HSV) thymidine kinase promoter. Using an internal control reporter allows to normalize the activity of the experimental reporter to the internal control, which minimizes experimental variability.