Localization of the Catalytic Domain of Copepod Luciferases: Analysis of Truncated Mutants of the Luciferase.

Luciferases from copepods and are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues.

Production of Copepod Luciferases via Baculovirus Expression System.

Secreted copepod luciferases (CopLucs) represent highly homologous enzymes which catalyze the oxidation of a low molecular weight substrate, coelenterazine, with the emission of blue light (λ = 485-488 nm), that is called bioluminescence (BL). The well-studied Gaussia (GLuc) and Metridia (MLuc) luciferases originally cloned from the marine copepods Gaussia princeps and Metridia longa belong to the group of the smallest natural luciferases. Their minimal molecular weight, high luminescent activity, cofactor-independent BL, and the ability to be secreted due to the own signal peptide open up the horizons for genetic engineering of CopLuc-based sensitive biosensors for in vivo imaging and in vitro analytical applications.

Production of Metridia Luciferase in Native Form by Oxidative Refolding from E. coli Inclusion Bodies.

The small coelenterazine-dependent luciferase from Metridia longa (MLuc), in view of its high activity, simplicity of bioluminescent (BL) reaction, and stability, has found successful analytical applications as a genetically encoded reporter for in vivo assessment of cellular processes. However, the study on the biochemical and BL properties and the development of in vitro analytical applications of MLuc are hampered by the difficulties of obtaining a sufficient amount of the highly active recombinant protein due to the presence of multiple (up to five) disulfide bonds per molecule. Here, we present a protocol to obtain the recombinant disulfide-rich MLuc using a cheap and simple Escherichia coli expression system without any affinity tags in its native form by refolding from inclusion bodies.

Crystal structure of semisynthetic obelin-v.

Coelenterazine-v (CTZ-v), a synthetic derivative with an additional benzyl ring, yields a bright bioluminescence of Renilla luciferase and its "yellow" mutant with a significant shift in the emission spectrum toward longer wavelengths, which makes it the substrate of choice for deep tissue imaging. Although Ca -regulated photoproteins activated with CTZ-v also display red-shifted light emission, in contrast to Renilla luciferase their bioluminescence activities are very low, which makes photoproteins activated by CTZ-v unusable for calcium imaging. Here, we report the crystal structure of Ca -regulated photoprotein obelin with 2-hydroperoxycoelenterazine-v (obelin-v) at 1.

The Smallest Isoform of Luciferase as a Fusion Partner for Hybrid Proteins.

Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod . In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner.

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications.

Copepod luciferases-a family of small secretory proteins of 18.4-24.3 kDa, including a signal peptide-are responsible for bright secreted bioluminescence of some marine copepods.

Bioluminescent and structural features of native folded Gaussia luciferase.

The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one-subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five SS bonds.

The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.

The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λ=480nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule.

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa.

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17-24 kDa) for M. longa luciferase have been cloned.

The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells.

The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M.

The smallest natural high-active luciferase: cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa.

Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.