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Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay [Text] / L. A. Frank [et al.] // Anal. Bioanal. Chem. - 2008. - Vol. 391, Is. 8. - P2891-2896, DOI 10.1007/s00216-008-2223-5. - Cited References: 22 . - ISSN 1618-2642

РУБ Biochemical Research Methods + Chemistry, Analytical
Рубрики:
ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   CRYSTAL-STRUCTURE
   BIOLUMINESCENCE
   AEQUORIN
   IMMUNOASSAY
   EXPRESSION
   CDNA
   PURIFICATION
   CLONING
Кл.слова (ненормированные):
Ca(2+)-regulated photoprotein -- bioluminescence -- dual-color assay
Аннотация: Two kinds of Ca(2+)-regulated photoprotein obelin with altered color of bioluminescence were obtained by active-center amino acid substitution. The mutant W92F-H22E emits violet light (lambda(max)=390 nm) and the mutant Y139F emits greenish light (lambda (max)=498 nm), with small spectral overlap, both display high activity and stability and thus may be used as reporters. For demonstration, the mutants were applied in dual-color simultaneous immunoassay of two gonadotropic hormones-follicle-stimulating hormone and luteinizing hormone. Bioluminescence of the reporters was simultaneously triggered by single injection of Ca(2+) solution, divided using band-pass optical filters and measured with a two-channel photometer. The sensitivity of simultaneous bioluminescence assay was close to that of a separate radioimmunoassay.

Держатели документа:
[Frank, Ludmila A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
[Frank, Ludmila A.
Borisova, Vasilisa V.
Markova, Svetlana V.
Malikova, Natalia P.
Stepanyuk, Galina A.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Borisova, V.V.; Markova, S.V.; Malikova, N.P.; Stepanyuk, G.A.; Vysotski, E.S.

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The smallest natural high-active luciferase: Cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa [Text] / S. V. Markova [et al.] // Biochem. Biophys. Res. Commun. - 2015. - Vol. 457, Is. 1. - P77-82, DOI 10.1016/j.bbrc.2014.12.082. - Cited References:20. - The cloning of cDNA encoding MLuc7 luciferase of M. longa was supported by Bayer AG (Germany); all other studies - by the grant 14-14-01119 of the Russian Science Foundation. We declare that authors have no conflict of interest. . - ISSN 0006-291X. - ISSN 1090-2104

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
CDNA CLONING
   SECRETED LUCIFERASE
   ESCHERICHIA-COLI
   EXPRESSION
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Copepod luciferase -- Mammalian -- expression -- Real-time imaging
Аннотация: 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 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 S-S bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of similar to 3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. (C) 2014 Elsevier Inc. All rights reserved.

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Держатели документа:
Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk, Russia.
Siberian Fed Univ, Chair Biophys, Krasnoyarsk, Russia.
ИБФ СО РАН

Доп.точки доступа:
Markova, Svetlana V.; Larionova, Marina D.; Burakova, Ludmila P.; Vysotski, Eugene S.; Bayer AG (Germany); Russian Science Foundation [14-14-01119]

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The Smallest Isoform ofMetridia longaLuciferase as a Fusion Partner for Hybrid Proteins / M. D. Larionova, S. V. Markova, N. V. Tikunova, E. S. Vysotski // Int. J. Mol. Sci. - 2020. - Vol. 21, Is. 14. - Ст. 4971, DOI 10.3390/ijms21144971. - Cited References:49. - The reported study was funded by the Russian Foundation for Basic Research (No. 18-44-242001), Government of Krasnoyarsk Territory, Krasnoyarsk Regional Fund of Science (S.V.M, M.D.L., and E.S.V.) and by the Russian State funded budget project of ICBFM SB RAS No. AAAA-A17-117020210027-9 (N.V.T.). . - ISSN 1422-0067

РУБ Biochemistry & Molecular Biology + Chemistry, Multidisciplinary
Рубрики:
COELENTERAZINE-BINDING PROTEIN
BIOLUMINESCENT REPORTER
GAUSSIA
Кл.слова (ненормированные):
bioluminescence -- coelenterazine -- copepod luciferase -- single-chain -- antibody -- immunoassay -- tick-borne encephalitis virus
Аннотация: 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 copepodMetridia longa. 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. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (K-D= 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (K-D= 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform ofM. longaluciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.

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Держатели документа:
Krasnoyarsk Sci Ctr SB RAS, Inst Biophys SB RAS, Photobiol Lab, Fed Res Ctr, Krasnoyarsk 660036, Russia.
Siberian Fed Univ, Sch Fundamental Biol & Biotechnol, Krasnoyarsk 660041, Russia.
Inst Chem Biol & Fundamental Med, Russian Acad Sci, Siberian Branch, Novosibirsk 630090, Russia.

Доп.точки доступа:
Larionova, Marina D.; Markova, Svetlana, V; Tikunova, Nina, V; Vysotski, Eugene S.; Vysotski, Eugene; Russian Foundation for Basic ResearchRussian Foundation for Basic Research (RFBR) [18-44-242001]; Krasnoyarsk Regional Fund of Science; ICBFM SB RAS [AAAA-A17-117020210027-9]; Government of Krasnoyarsk Territory

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The smallest isoform of Metridia longa luciferase as a fusion partner for hybrid proteins / M. D. Larionova, S. V. Markova, N. V. Tikunova, E. S. Vysotski // Int. J. Mol. Sci. - 2020. - Vol. 21, Is. 14. - Ст. 4971. - P1-16, DOI 10.3390/ijms21144971 . - ISSN 1661-6596
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Copepod luciferase -- Immunoassay -- Single-chain antibody -- Tick-borne encephalitis virus -- fusion protein -- glycoprotein -- histidine -- messenger RNA -- Metridia longa luciferase -- recombinant protein -- single chain fragment variable antibody -- unclassified drug -- amino terminal sequence -- antibody affinity -- antigen binding -- Article -- binding assay -- binding site -- bioluminescence -- bioluminescence resonance energy transfer -- cross reaction -- dissociation constant -- enzyme activity -- Escherichia coli -- gene -- genetic engineering -- genetic transfection -- immunoassay -- limit of detection -- mluc7 gene -- molecular cloning -- nonhuman -- nucleotide sequence -- protein expression -- protein purification -- protein unfolding -- spectral sensitivity -- tick borne encephalitis -- Tick borne encephalitis virus
Аннотация: 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 Metridia longa. 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. We demonstrate that, whereas fusion of a single-chain antibody to either N-or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Scopus
Держатели документа:
Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, 660036, Russian Federation
School of Fundamental Biology and Biotechnology, Siberian Federal University, Krasnoyarsk, 660041, Russian Federation
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090, Russian Federation

Доп.точки доступа:
Larionova, M. D.; Markova, S. V.; Tikunova, N. V.; Vysotski, E. S.

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The photoprotein obelin as a bioluminescent reporter to monitor protein-protein interactions in vivo and in vitro by protein-fragment complementation assays [Text] / S. V. Markova, P. V. Natashin, E. S. Vysotski // J. Biotechnol. - 2010. - Vol. 150: 14th International Biotechnology Symposium and Exhibition (IBS-2008) (SEP 14-18, 2010, Rimini, ITALY). - S93-S93, DOI 10.1016/j.jbiotec.2010.08.241. - Cited References: 0 . - ISSN 0168-1656

РУБ Biotechnology & Applied Microbiology

Кл.слова (ненормированные):
Bioluminescence -- Reporter -- Obelin -- Protein-protein interactions

Держатели документа:
[Markova, S. V.
Vysotski, E. S.] Russian Acad Sci, Inst Biophys, Siberian Branch, Moscow 117901, Russia
[Markova, S. V.
Natashin, P. V.] Siberian Fed Univ, Krasnoyarsk, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Natashin, P.V.; Vysotski, E.S.

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The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells / M. D. Larionova, S. V. Markova, E. S. Vysotski // Biochem. Biophys. Res. Commun. - 2017. - Vol. 483, Is. 1. - P772-778, DOI 10.1016/j.bbrc.2016.12.067. - Cited References:24. - The cloning of cDNAs encoding MLuc2 isoforms of M. longa was supported by Bayer AG (Germany) and the state budget allocated to the fundamental research at the Russian Academy of Sciences (project No. 01201351504); all other studies were funded by RFBR and Government of Krasnoyarsk Territory according to the research project No. 16-44-242099. . - ISSN 0006-291X. - ISSN 1090-2104

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
CDNA CLONING
EXPRESSION
ENZYME
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Bioluminescent reporter -- Psychrophilic -- enzyme -- Molecular adaptation
Аннотация: The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazinedependent 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. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only similar to 54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/ insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 degrees C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at similar to 5 degrees C and 1 M NaCl. The MLuc(2) adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations. (C) 2016 Elsevier Inc. All rights reserved.

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Держатели документа:
Fed Res Ctr Krasnoyarsk Sci Ctr SB RAS, Inst Biophys SB RAS, Photobiol Lab, Krasnoyarsk, Russia.
Siberian Fed Univ, Krasnoyarsk, Russia.

Доп.точки доступа:
Larionova, Marina D.; Markova, Svetlana V.; Vysotski, Eugene S.; Bayer AG (Germany); Russian Academy of Sciences [01201351504]; RFBR; Government of Krasnoyarsk Territory [16-44-242099]

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The Ca2+-Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation / V. V. Krasitskaya, N. S. Goncharova, V. V. Biriukov [et al.] // Photochem. Photobiol. - 2020, DOI 10.1111/php.13274. - Cited References:25. - This work has been supported by the Russian Foundation for Basic Research (RFBR) under the grant no 18-38-00531. . - Article in press. - ISSN 0031-8655. - ISSN 1751-1097

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
CARDIAC TROPONIN-I
   BIOLUMINESCENCE
   IMMUNOASSAY
   APTASENSOR
   DIAGNOSIS
Аннотация: Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer-target complexes during design and development of the specific aptamers. The assay involves Ca2+-regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.

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Держатели документа:
Fed Res Ctr KSC SB RAS, Inst Biophys SB RAS, Krasnoyarsk, Russia.
Siberian Fed Univ, Krasnoyarsk, Russia.
Inst Chem Biol & Fundamental Med SB RAS, Novosibirsk, Russia.
Fed Res Ctr KSC SB RAS, Kirensky Inst Phys, Krasnoyarsk, Russia.

Доп.точки доступа:
Krasitskaya, Vasilisa V.; Goncharova, Natalia S.; Biriukov, Vladislav V.; Bashmakova, Eugenia E.; Kabilov, Marsel R.; Baykov, Ivan K.; Sokolov, Aleksey E.; Frank, Ludmila A.; Russian Foundation for Basic Research (RFBR)Russian Foundation for Basic Research (RFBR) [18-38-00531]

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Structural distinctions of fast and slow bacterial luciferases revealed by phylogenetic analysis [Text] / A. A. Deeva [et al.] // Bioinformatics. - 2016. - Vol. 32, Is. 20. - P3053-3057, DOI 10.1093/bioinformatics/btw386. - Cited References:31. - The reported study was partially funded by RFBR according to the research project No. 16-34-00746 mol_a; by the Ministry of Education and Science of the Russian Federation [project No 1762] and by the state budget allocated to the fundamental research at the Russian Academy of Sciences [project No 01201351504]. . - ISSN 1367-4803. - ISSN 1460-2059

РУБ Biochemical Research Methods + Biotechnology & Applied Microbiology

Аннотация: Motivation: Bacterial luciferases are heterodimeric enzymes that catalyze a chemical reaction, so called bioluminescence, which causes light emission in bacteria. Bioluminescence is vastly used as a reporter system in research tools and commercial developments. However, the details of the mechanisms that stabilize and transform the reaction intermediates as well as differences in the enzymatic kinetics amongst different bacterial luciferases remain to be elucidated. Results: Amino acid sequences alignments for 21 bacterial luciferases (both alpha- and beta-subunits) were analyzed. For alpha-subunit, containing the enzyme active center, 48 polymorphic amino acid positions were identified. According to them, the sequences fell into two distinct groups known as slow and fast based on the decay rate of the bioluminescence reaction. The differences in the enzyme active site induced by structural polymorphism are analyzed.

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Scopus
Держатели документа:
Siberian Fed Univ, Lab Bioluminescent Biotechnol, Krasnoyarsk, Russia.
Inst Cell Biophys RAS, Mech Cell Genome Functioning Lab, Pushchino, Moscow Region, Russia.
Moscow Inst Phys & Technol, Dolgoprudnyi, Russia.
Inst Biophys SB RAS, Lab Photobiol, Krasnoyarsk, Russia.

Доп.точки доступа:
Deeva, Anna A.; Temlyakova, Evgenia A.; Sorokin, Anatoly A.; Nemtseva, Elena V.; Kratasyuk, Valentina A.; RFBR [16-34-00746 mol_a]; Ministry of Education and Science of the Russian Federation [1762]; state budget allocated to the fundamental research at Russian Academy of Sciences [01201351504]

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Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications. / S. V. Markova, M. D. Larionova, E. S. Vysotski // Photochemistry and photobiology. - 2018, DOI 10.1111/php.13077 . - ISSN 1751-1097
Аннотация: 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. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine-dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high-throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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Держатели документа:
Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center "Krasnoyarsk Science Center SB RAS", Krasnoyarsk, 660036, Russia.
Siberian Federal University, Krasnoyarsk, 660041, Russia.
N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia, Moscow, 115478, Russia.

Доп.точки доступа:
Markova, Svetlana V.; Larionova, Marina D.; Vysotski, Eugene S.

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10.

Reporter-recruiting bifunctional aptasensor for bioluminescent analytical assays / A. Davydova, V. Krasitskaya, P. Vorobjev [et al.] // RSC Adv. - 2020. - Vol. 10, Is. 54. - P32393-32399, DOI 10.1039/d0ra05117a. - Cited References:33. - The work was supported by the Russian Science Foundation (grant #16-14-10296), Russian State funded budget projects #AAAA-A17-117020210021-7 to ICBFM SB RAS and #AAAA-A19-119031890015-0 to IBP SB RAS. . - ISSN 2046-2069

РУБ Chemistry, Multidisciplinary
Рубрики:
DNA APTAMER
   RNA APTAMER
   OBELIN
   PURIFICATION
   EXPRESSION
   SEQUENCES
Аннотация: We report a novel bioluminescent aptasensor, which consists of 2 '-F-RNA aptamer modules joined into a bi-specific aptamer construct. One aptamer module binds the analyte, then after structural rearrangement the second module recruits non-covalently Ca2+-dependent photoprotein obelin from the solution, thus providing a bioluminescent signal. This concept allows using free protein as a reporter, which brings such advantages as no need for aptamer-protein conjugation, a possibility of thermal re-folding of aptamer component with no harm to a protein, and simpler detection protocol. We developed the new 2 '-F-RNA aptamer for obelin, and proposed the strategy for engineering structure-switching bi-modular aptamer constructs which bind the analyte and the obelin in a sequential manner. With the use of hemoglobin as a model analyte, we showed the feasibility of utilizing the aptasensor in a fast and straightforward bioluminescent microplate assay. With a proper design of a secondary structure, this strategy of aptasensor engineering might be further extended to bi-specific aptamer-based bioluminescent sensors for other analytes of interest.

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Держатели документа:
SB RAS, Inst Chem Biol & Fundamental Med, Novosibirsk 630090, Russia.
SB RAS, Inst Biophys, Fed Res Ctr, Krasnoyarsk Sci Ctr, Krasnoyarsk 660036, Russia.
Novosibirsk State Univ, Pimgova St 2, Novosibirsk 630090, Russia.
Siberian Fed Univ, Krasnoyarsk 660041, Russia.

Доп.точки доступа:
Davydova, Anna; Krasitskaya, Vasilisa; Vorobjev, Pavel; Timoshenko, Valentina; Tupikin, Alexey; Kabilov, Marsel; Frank, Ludmila; Venyaminova, Alya; Vorobyeva, Mariya; Russian Science FoundationRussian Science Foundation (RSF) [16-14-10296]; Russian State funded budget projects [AAAA-A17-117020210021-7, AAAA-A19-119031890015-0]

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11.

Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay [Text] / V. V. Borisova [et al.] // Photochem. Photobiol. Sci. - 2008. - Vol. 7, Is. 9. - P1025-1031, DOI 10.1039/b807271j. - Cited References: 19. - The work was supported by Bayer AG, by the Russian Foundation for Basic Research grants 05-04-48271 and 06-04-08076, by the joint grant 06-04-89502 of the Russian Foundation for Basic Research and Taiwan National Science Council, and by the "Molecular and Cellular Biology" program of the Russian Academy of Sciences. . - ISSN 1474-905X

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical
Рубрики:
BIOLUMINESCENT REPORTER
   GAUSSIA LUCIFERASE
   CDNA
   PROTEINS
   CLONING
   OVEREXPRESSION
   PURIFICATION
   MUTAGENESIS
   ENZYME
   OBELIN
Аннотация: The recombinant coelenterazine-dependent luciferases (isoforms MLuc 164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc09) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40% of the initial activity The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.

Держатели документа:
[Borisova, Vasillisa V.
Frank, Ludmila A.
Markova, Svetlana V.
Burakova, Ludmilla P.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
[Frank, Ludmila A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Borisova, V.V.; Frank, L.A.; Markova, S.V.; Burakova, L.P.; Vysotski, E.S.

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12.

Obelin as a carrier protein and reporter enzyme for in vitro synthesized small bioactive polypeptides: New approach to obtain sarcotoxin [Text] / B. A. Illarionov [et al.] ; ed.: JW Hastings, LJ Kricka, J Kricka, // BIOLUMINESCENCE AND CHEMILUMINESCENCE: MOLECULAR REPORTING WITH PHOTONS : JOHN WILEY & SONS LTD, 1997. - 9th International Symposium on Bioluminescence and Chemiluminescence (OCT , 1996, WOODS HOLE, MA). - P435-438. - Cited References: 0 . - ISBN 0-471-97502-8

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical

: 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Illarionov, B.A.; Matveev, S.V.; Bondar, V.S.; Skosyrev, V.A.; Alakhov, Y.B.; Hastings, JW \ed.\; Kricka, LJ \ed.\; Kricka,, J \ed.\

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13.

Obelin as a carrier protein and reporter enzyme for in vitro synthesized small bioactive polypeptides: New approach to obtain sarcotoxin [Text] / B. A. Illarionov [et al.] ; ed.: JW Hastings, LJ Kricka, J Kricka, // BIOLUMINESCENCE AND CHEMILUMINESCENCE: MOLECULAR REPORTING WITH PHOTONS : JOHN WILEY & SONS LTD, 1997. - 9th International Symposium on Bioluminescence and Chemiluminescence (OCT , 1996, WOODS HOLE, MA). - P. 435-438. - Cited References: 0 . - ISBN 0-471-97502-8

РУБ Biochemistry & Molecular Biology + Biophysics + Chemistry, Physical

WOS : 660036, Красноярск, Академгородок, д. 50, стр. 50
Доп.точки доступа:
Illarionov, B.A.; Matveev, S.V.; Bondar, V.S.; Skosyrev, V.A.; Alakhov, Y.B.; Hastings, JW \ed.\; Kricka, LJ \ed.\; Kricka,, J \ed.\

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14.

Large-scale, high-throughput validation of short hairpin RNA sequences for RNA interference [Text] / L. H. Lamarcq [et al.] // J. Biomol. Screen. - 2006. - Vol. 11, Is. 3. - P236-246, DOI 10.1177/1087057105284342. - Cited References: 50 . - ISSN 1087-0571

РУБ Biochemical Research Methods + Biotechnology & Applied Microbiology + Chemistry, Analytical
Рубрики:
ENZYME FRAGMENT COMPLEMENTATION
   ORFEOME VERSION 1.1
   SMALL NUCLEAR-RNA
   MAMMALIAN-CELLS
   GENE-EXPRESSION
   SIRNA SEQUENCES
   POLYMERASE-III
   SELECTION
   TRANSCRIPTION
   MICROARRAYS
Кл.слова (ненормированные):
RNAi -- high-throughput screening -- target validation -- shRNA -- reporter assay
Аннотация: (shRNAs) is described. Using this approach, 464 shRNAs auainst 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAS against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position-specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).

Держатели документа:
Clontech Labs, Mountain View, CA 94043 USA
BD Biosci Pharmingen, La Jolla, CA USA
Bayer Healthcare AG, Pharma Res, Mol Screening Technol, Wuppertal, Germany
Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Lamarcq, L.H.; Scherer, B.J.; Phelan, M.L.; Kalnine, N.N.; Nguyen, Y.H.; Kabakova, T...; Chen, X.Y.; Tan, M...; Chang, C...; Berlon, C...; Campos-Gonzalez, R...; Gao, G.J.; Golz, S...; Vysotski, E.S.; Farmer, A.A.

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15.

Interchange of aequorin and obelin bioluminescence color is determined by substitution of one active site residue of each photoprotein [Text] / G. A. Stepanyuk [et al.] // FEBS Lett. - 2005. - Vol. 579, Is. 5. - P1008-1014, DOI 10.1016/j.febslet.2005.01.004. - Cited References: 49 . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
FIREFLY LUCIFERASE
   SEQUENCE-ANALYSIS
   CA2+-REGULATED PHOTOPROTEINS
   CA2+-DISCHARGED PHOTOPROTEIN
   VIOLET BIOLUMINESCENCE
   INTRACELLULAR CALCIUM
   ENDOPLASMIC-RETICULUM
   ANGSTROM RESOLUTION
   CRYSTAL-STRUCTURE
   APOAEQUORIN CDNA
Кл.слова (ненормированные):
coelenterazine -- calcium -- reporter protein -- mammalian expression -- fluorescence spectrum
Аннотация: The bioluminescence spectra from the Ca2+-regulated photoproteins aequorin (lambda(max) = 469 nm) and obelin (lambda(max) = 482 nm) differ because aequorin has an H-bond from its Tyr82 to the bound coelenteramide, not present in obelin at the corresponding Phe88. Substitutions of this Phe88 by Tyr, Trp, or His shifted the obelin bioluminescence to shorter wavelength with F88Y having lambda(max) = 453 nm. Removal of the H-bond by the substitution of Y82F in aequorin shifted its bioluminescence to lambda(max) = 501 nm. All mutants were stable with good activity and were expressible in mammalian cells, thereby demonstrating potential for monitoring multiple events in cells using multi-color detection. (C) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
Bayer AG, Pharma Res Mol Screening Technol, D-42096 Wuppertal, Germany
Univ Georgia, Dept Mol Biol & Biochem, Athens, GA 30602 USA
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Stepanyuk, G.A.; Golz, S...; Markova, S.V.; Frank, L.A.; Lee, J...; Vysotski, E.S.

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16.

17.

Hybrid Minimal Core Streptavidin–Obelin as a Versatile Reporter for Bioluminescence-based Bioassay / E. E. Bashmakova [et al.] // Photochem. Photobiol. - 2017. - Vol. 93, Is. 2. - P548-552, DOI 10.1111/php.12648 . - ISSN 0031-8655
Аннотация: Ca2+-regulated photoprotein obelin was genetically fused with a minimum-sized core streptavidin. Hybrid protein (SAV–OL) was produced by bacterial expression and applied as a specific bioluminescent probe in diverse solid-phase assays. The obtained results clearly demonstrate specific activity of each domain indicating its proper folding with favorable space orientation. SAV–OL has been shown to be a much more sensitive label than the chemical conjugate of a full-length streptavidin with obelin. © 2016 The American Society of Photobiology

Scopus,
Смотреть статью,
WOS
Держатели документа:
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russian Federation
Siberian Federal University, Krasnoyarsk, Russian Federation
Chemistry Department, Lomonosov Moscow State University, Moscow, Russian Federation

Доп.точки доступа:
Bashmakova, E. E.; Krasitskaya, V. V.; Kudryavtsev, A. N.; Grigorenko, V. G.; Frank, L. A.

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18.

Highly active BRET-reporter based on yellow mutant of Renilla muelleri luciferase / E. V. Eremeeva, S. V. Markova, E. S. Vysotski // Dokl. Biochem. Biophys. - 2013. - Vol. 450, Is. 1. - P147-150, DOI 10.1134/S1607672913030095. - Cited References: 14. - This work was supported by the Ministry of Education and Science of the Russian Federation (Government Contract no. 16.512.11.2141) and Council of the President of the Russian Federation on Grants and State Support of Leading Scientific Schools (project no. NSh-64987.2010.4). . - ISSN 1607-6729

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
GREEN-FLUORESCENT PROTEIN
   GENE-EXPRESSION
   CDNA
   CLONING
   BIOLUMINESCENCE
   RENIFORMIS

Scopus
Держатели документа:
[Eremeeva, E. V.
Markova, S. V.
Vysotski, E. S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Krasnoyarsk 660036, Russia
[Eremeeva, E. V.
Markova, S. V.
Vysotski, E. S.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
ИБФ СО РАН
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036, Russian Federation
Siberian Federal University, Svobodnyi pr. 79, Krasnoyarsk, 660041, Russian Federation : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Vysotski, E.S.

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19.

High-active truncated luciferase of copepod Metridia longa / S. V. Markova, L. P. Burakova, E. S. Vysotski // Biochem. Biophys. Res. Commun. - 2012. - Vol. 417, Is. 1. - P98-103, DOI 10.1016/j.bbrc.2011.11.063. - Cited References: 31. - This study was supported by the Grants 16.512.11.2141 and 64987.2010.4 of the Ministry of Education and Science of Russian Federation. . - ISSN 0006-291X

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
COELENTERAZINE-BINDING PROTEIN
   REPORTER-GENE-EXPRESSION
   RENILLA LUCIFERASE
   GAUSSIA LUCIFERASE
   LIGHT-EMITTER
   IN-VIVO
   BIOLUMINESCENCE
   PHOTOPROTEINS
   CDNA
   SUBSTRATE
Кл.слова (ненормированные):
Bioluminescence -- Coelenterazine -- Mammalian expression -- Secretion
Аннотация: The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coil cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme. (C) 2011 Elsevier Inc. All rights reserved.

Держатели документа:
[Vysotski, Eugene S.] Russian Acad Sci, Photobiol Lab, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Russia
Siberian Fed Univ, Dept Biophys, Krasnoyarsk 660041, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Markova, S.V.; Burakova, L.P.; Vysotski, E.S.

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20.

FOXC1-Mediated Effects of miR-204-5p on Melanoma Cell Proliferation / I. Y. Dubovtseva, M. B. Aksenenko, E. D. Nikolaeva [и др.] // Mol Biol (Mosk). - 2021. - Vol. 55, Is. 4. - С. 667-675, DOI 10.31857/S0026898421030058 . - ISSN 0026-8984
Кл.слова (ненормированные):
BRO -- dormant cancer cells -- FOXC1 -- melanoma -- miR-204-5p -- miRNA -- siRNA -- SK-MEL-2 -- forkhead transcription factor -- FOXC1 protein, human -- microRNA -- MIRN204 microRNA, human -- cell motion -- cell proliferation -- genetics -- human -- melanoma -- tumor cell line -- Cell Line, Tumor -- Cell Movement -- Cell Proliferation -- Forkhead Transcription Factors -- Humans -- Melanoma -- MicroRNAs
Аннотация: MicroRNAs epigenetically regulate physiological and pathological processes. Previously, we found that miR-204-5p is expressed at low levels in melanoma cells, and an increase in its level leads to a change in proliferation, migration, and invasion of these cancer cells. Now, using bioinformatics analysis, it has been shown that the target of miR-204-5p is FOXC1 transcription factor, which is implicated in carcinogenesis. Using the luciferase reporter assay, it was found that miR-204-5p suppresses expression of the FOXC1 gene by binding to its 3' non-coding region. Transfection of small interfering RNA (siRNA) targeting FOXC1 into melanoma cells caused a decrease in miR-204-5p levels, which is consistent with the generally accepted concept of feedback regulation of miRNA expression by target genes. According to the results of the MTT test and fluorescence microscopy, the proliferation level of melanoma cells under the influence of siRNA to FOXC1 decreased 72 h after transfection. Changes in the ratio of cells by cell cycle phase were analyzed using flow cytometry. Regulatory relationships between FOXC1 and miR-204-5p, and an inhibitory effect of FOXC1 knockdown on melanoma cell proliferation were revealed. Based on the results, it can be assumed that miR-204-5p regulates proliferation of melanoma cells by affecting FOXC1 expression.

Scopus
Держатели документа:
Voino-Yasenetsky Krasnoyarsk State Medical University, Ministry of Health of the Russian Federation, Krasnoyarsk, 660022, Russian Federation
Siberian Branch of the Russian Academy of Sciences, Research Institute for Medical Problems in the North, Krasnoyarsk, 660022, Russian Federation
Biophysics Institute of the Siberian Branch of the RAS - Division of Federal Research Center "Krasnoyarsk Scientific Center of the Siberian Branch of the RAS", Krasnoyarsk, 660022, Russian Federation

Доп.точки доступа:
Dubovtseva, I. Y.; Aksenenko, M. B.; Nikolaeva, E. D.; Averchuk, A. S.; Moshev, A. V.; Savchenko, A. A.; Markova, S. V.; Ruksha, T. G.

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