Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator

Illarionov, Boris; Frank, Ludmila A.; Illarionova, Victoria; Bondar, V. S.; Vysotski, Eugene S.; Blinks, John R.

doi:10.1016/s0076-6879(00)05491-4

Cited by 91 publications

(100 citation statements)

References 36 publications

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“…Findings obtained from this research have many practical applications (Morin 1974, Blinks et al 1976, 1982, Ugarova & Brovko 1981, Gitelson et al 1984, Ugarova et al 1987, 1993, Illarionov et al 2000Shimomura 2006;Girotti et al 2008, Medvedeva et al 2009, Fernandez-Pinas et al 2014, Markova & Vysotski 2015.…”

Section: Introductionmentioning

confidence: 84%

Luminescence of cold extracts from mycelium of luminous basidiomycetes during long–term storage

Puzyr¹

2017

CREAM

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Cold extracts with high activities of enzymes of luminescent reaction were prepared from mycelia of luminous fungi Armillaria borealis IBSO 2328, Mycena citricolor IBSO 2331, and Neonothopanus nambi IBSO 2391. The authors describe techniques of preparing cold extracts with high levels of luminescence from mycelial biomass of different species of luminous basidiomycetes. The investigation of cold extracts showed that in experiments with freezing and thawing of the samples as well as in experiments with lyophilization followed by dissolution of the dry samples, the levels of enzyme activity were high, with in vitro luminescence exhibited after addition of NADPH and the hot extract containing the substrate. High activity levels of the enzymes of luminescent reaction were measured in lyophilized cold extracts stored over three years. In lyophilized preparations, the enzymes of luminescent reaction had high thermostability, even when dry preparations of cold extracts were exposed to a temperature of 100°C for 60 minutes.

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Section: Introductionmentioning

confidence: 84%

Luminescence of cold extracts from mycelium of luminous basidiomycetes during long–term storage

Puzyr¹

2017

CREAM

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“…mCherry-obelin was cloned between the NheI and XbaI sites of pcDNA3.1/zeo(1). The obelin biosensor consists of an amino-terminal-mCherry joined by 5 amino acid residue linker (GSAGT) to the obelin calcium-activated photoprotein (Illarionov et al, 2000). The obelin biosensor is a bioluminescent photoprotein derived from Obelia longissima that tightly binds the chromophore (coelenterazine) with oxygen to form a stable complex, which is activated upon binding 3 calcium ions (linear range of sensitivity: 100 nM-100 mM; Illarionov et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were preincubated with the obelin substrate, coelenterazine cp (1 mM; 25°C), for 2 hours in the dark. Under basal conditions, a low level of calcium-independent luminescence is observed (Illarionov et al, 2000). Upon calcium binding, the photoprotein oxidizes coelenterazine cp, converting it to coelenteramide, releasing carbon dioxide and light in the blue range (465-495 nm) (Illarionov et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

“…Under basal conditions, a low level of calcium-independent luminescence is observed (Illarionov et al, 2000). Upon calcium binding, the photoprotein oxidizes coelenterazine cp, converting it to coelenteramide, releasing carbon dioxide and light in the blue range (465-495 nm) (Illarionov et al, 2000). Therefore, with an increase in intracellular calcium levels, an increase in luminescence is observed.…”

Section: Methodsmentioning

confidence: 99%

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Quantification of Ligand Bias for Clinically Relevant β₂-Adrenergic Receptor Ligands: Implications for Drug Taxonomy

et al. 2013

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The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the b2-adrenergic receptor as a model, we present a proof of concept study that assessed the bias of 19 b-adrenergic ligands, including many clinically used compounds, across four pathways [cAMP production, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, calcium mobilization, and receptor endocytosis] in the same cell background (human embryonic kidney 293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from offtarget effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the nonselective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptoreffector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signaling profiles, drug classification, therapeutic efficacy, and safety.

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“…conditions. So a practically unlimited source of highly pure recombinant obelin becomes available (Illarionov et al, 2000). Obelin was shown to be stable to chemical modifications and its conjugates with biospecific moleculesimmunoglobulins, haptens, oligonucleotides, etc., were obtained and successfully applied as labels in binding assay (Frank et al, 2004b).…”

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confidence: 99%

Genetically Modified Coelenterazine-Dependent Luciferases as Reporters for In Vitro Assay

Krasitskaya¹,

Kudryavtsev²,

Frank³

et al. 2017

J. Sib. Fed. Univ., Biol.

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Among the most important tasks of modern biotechnology is the development of new analytical techniques suitable for highly sensitive and specific detection of diagnostically important targets rapidlyexpanding their range with the progress in biomedical science. In this view, particular attention is paid to elaboration of methods based on photosensitive technologies (fluorescent, chemiluminescent, etc.) Apart from fundamental research, considerable efforts have been made with regard to analytical applications of those in vitro (Frank et al., 1997(Frank et al., , 2004a(Frank et al., , 2007. The important moment here is that a high quantum yield of the reactions catalyzed by luciferases and a high signal-to-noise ratio provide high sensitivity of the assays based on these enzymes as reporters.

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Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator

Cited by 91 publications

References 36 publications

Luminescence of cold extracts from mycelium of luminous basidiomycetes during long–term storage

Luminescence of cold extracts from mycelium of luminous basidiomycetes during long–term storage

Quantification of Ligand Bias for Clinically Relevant β₂-Adrenergic Receptor Ligands: Implications for Drug Taxonomy

Genetically Modified Coelenterazine-Dependent Luciferases as Reporters for In Vitro Assay

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Recombinant obelin: Cloning and expression of cDNA, purification, and characterization as a calcium indicator

Cited by 91 publications

References 36 publications

Luminescence of cold extracts from mycelium of luminous basidiomycetes during long–term storage

Luminescence of cold extracts from mycelium of luminous basidiomycetes during long–term storage

Quantification of Ligand Bias for Clinically Relevant β2-Adrenergic Receptor Ligands: Implications for Drug Taxonomy

Genetically Modified Coelenterazine-Dependent Luciferases as Reporters for In Vitro Assay

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Product

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Quantification of Ligand Bias for Clinically Relevant β₂-Adrenergic Receptor Ligands: Implications for Drug Taxonomy