Preparation
Fireflies, the raw material for FL production, rely on artificial capture or breeding, which is limited by geographical and seasonal factors. Furthermore, the production cycle of this organism is long, breeding costs are high, and the extracted enzyme composition is complex. With the development of molecular biology, researchers have turned to genetic engineering methods for production. In 1985, deWet, JR, et al. first cloned the FL gene of *P. pyralis* and expressed it in *Escherichia coli*, obtaining active FL. In 1986, they determined the cDNA sequence of the FL gene. Subsequently, the FL genes of various bioluminescent beetles were successfully cloned and expressed in prokaryotic and eukaryotic expression systems. The cDNA gene from *P. pyralis*, approximately 1.8 kb long, expressed in *E. coli* produces a polypeptide chain with a molecular weight of 62 kDa and containing 550 amino acids. Compared with naturally extracted FL, it has the same reaction kinetics and emission wavelength, and the enzyme stability is also identical under the same reaction conditions. The firefly luciferase gene was cloned and expressed in *E. coli*. Transformants were grown at 37°C in LB medium until the late logarithmic phase, at which point the cells were collected. Cytoplasmic components were extracted using osmotic pressure and freeze-thaw methods. After homogenization and centrifugation, the purified enzyme was extracted using gel size exclusion and ion-exchange chromatography and then freeze-dried for preservation. BL can be directly extracted from cultured luminescent bacteria, and its preparation method is similar to that of FL. The enzyme activity extracted from *V. harvestyi* can reach 1.8 × 10¹¹ LU/mg. For specific applications, BL can also be obtained through mutagenesis or gene cloning. Numerous reports exist on gene cloning of various luminescent bacteria.
Description
Luciferase (firefly recombinant) is a light-generating enzyme that catalyzes an oxidative reaction, using luciferin, ATP, and molecular oxygen to produce oxyluciferin, which yields visible light. The bioluminescence of this ATP-dependent luciferase is commonly used in reporter systems that are amenable to high-throughput screening.
Uses
Firefly luciferase is used extensively in molecular and cell biology, in particular for the efficient detection and quantitation of ATP and as a reporter for genetic function.
Uses
Luciferase from
Photinus pyralis (firefly) has been used as a component of lysis solution to measure luminescence signals, as part of the study to determine chemical signals which can activate the karrikin insensitive 2 (KAI2) pathway.
General Description
Firefly luciferase catalyzes the reaction of luciferin with ATP and leads to the production of yellow-green light. The enzyme has a molecular weight of 62kDa and is expressed in peroxisomes. Luciferase is considered as a model to study protein–anesthetic interactions. Firefly luciferase is highly useful in cell biology and molecular biology, as a reporter of gene function and for the quantification of ATP.
Biochem/physiol Actions
Firefly luciferase is a 62 kDa protein that catalyzes the production of light. The enzyme requires ATP, molecular oxygen, and the heterocyclic compound, firefly luciferin, to generate light in a two-step process. The light producing reaction is initiated by luciferin activation (adenylation of its carboxylate group) and proceeds in the presence of molecular oxygen to yield a photon of yellow-green light.
Description
Luciferase produced in E.Coli is a single, non-glycosylated polypeptide chain containing 571 amino acids (1-550 a.a.) and having a molecular mass of 62.9kDa.
Luciferase is fused to a 21 amino acid His-tag at N-terminus & purified by proprietary chromatographic techniques.
Background
Luciferase is a general term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. Luciferase catalyzes a bioluminescent reaction which involves the substrate luciferin as well as Mg2+ and ATP, produces green light with a wavelength of 562 nm. Luciferase from firefly is broadly used as a reporter for studying gene regulation and function, and for pharmaceutical screening.
References
[1] BALDWIN T O. Firefly luciferase: the structure is known, but the mystery remains.[J]. Structure, 1996, 4 3: 223-228. DOI:
10.1016/s0969-2126(96)00026-3[2] TINGTING XU. The Expanding Toolbox of In Vivo Bioluminescent Imaging.[J]. Frontiers in Oncology, 2016: 150. DOI:
10.3389/fonc.2016.00150[3] De Souza, F.I., Zumiotti, A.V., and Da Silva, C.F. Neuregulins 1-α and 1-β on the regeneration the peripheral nerves[J]. Acta Ortop Bras.
