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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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Use of proZZ-obelin fusion protein in bioluminescent immunoassay [Text] / L. A. Frank, V. A. Illarionova, E. S. Vysotski // Biochem. Biophys. Res. Commun. - 1996. - Vol. 219, Is. 2. - P475-479, DOI 10.1006/bbrc.1996.0258. - Cited References: 21 . - 5. - ISSN 0006-291X

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
ESCHERICHIA-COLI
   EXPRESSION
   AEQUORIN
   PURIFICATION
   SYSTEM
Аннотация: Obelin is a photoprotein that emits light by Ca2+-binding. To develop a bioluminescent immunoassay based on the light emission property of obelin, we have expressed the apoobelin fusion protein with ZZ-domain of S. aureus protein A in E. coil by recombinant DNA techniques. The pro2Z-obelin expressed was purified by one-step affinity chromatography on IgG-Agarose. The purified proZZ-obelin has both the luminescent activity of obelin and the IgG-binding ability of ZZ-domain. The specific activity of fusion protein was 8.5 x 10(15) photons per mg of protein. (C) 1996 Academic Press, Inc.
: 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Frank, L.A.; Illarionova, V.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 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 Recombinant Luciferase of the Fungus Neonothopanus nambi: Obtaining and Properties / A. Y. Gorokhovatsky, T. V. Chepurnykh, A. S. Shcheglov [et al.] // Doklad. Biochem. Biophys. - 2021. - Vol. 496, Is. 1. - P52-55, DOI 10.1134/S1607672921010051 . - ISSN 1607-6729
Кл.слова (ненормированные):
bioluminescence -- heterologous expression -- luciferase -- Neonothopanus nambi -- nnLuz -- Pichia pastoris
Аннотация: Abstract: A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase. © 2021, The Author(s),.

Scopus
Держатели документа:
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation
Institute of Biophysics, Federal Research Center “Krasnoyarsk Scientific Center of the Siberian Branch of the Russian Academy of Sciences”, Krasnoyarsk, Russian Federation

Доп.точки доступа:
Gorokhovatsky, A. Y.; Chepurnykh, T. V.; Shcheglov, A. S.; Mokrushina, Y. A.; Baranova, M. N.; Goncharuk, S. A.; Purtov, K. V.; Petushkov, V. N.; Rodionova, N. S.; Yampolsky, I. V.

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The Recombinant Luciferase of the Fungus Neonothopanus nambi: Obtaining and Properties / A. Y. Gorokhovatsky, T. V. Chepurnykh, A. S. Shcheglov [et al.] // Dokl. Biochem. Biophys. - 2021. - Vol. 496, Is. 1. - P52-55, DOI 10.1134/S1607672921010051. - Cited References:10. - The work was supported by the Russian Science Foundation (project no. 16-14-00052-P). The creation of the luciferase-producing yeast strain nnLuz was supported by the President's grant for state support of the leading scientific schools of the Russian Federation (NSh-2605.2020.4). . - ISSN 1607-6729. - ISSN 1608-3091

РУБ Biochemistry & Molecular Biology + Biophysics

Кл.слова (ненормированные):
bioluminescence -- luciferase -- nnLuz -- Neonothopanus nambi -- heterologous -- expression -- Pichia pastoris
Аннотация: A key component of the recently described bioluminescent system of higher fungi is luciferase, a new class of proteins. The properties of fungal luciferase and their relationship with its structure are interesting both for improving autoluminescent systems already created on its basis and for creating new ones. Therefore, it is extremely important to understand the spatial structure of this protein. We have performed heterologous expression and purification of Neonothopanus nambi luciferase, obtained a protein suitable for subsequent crystallization, and also determined some biochemical properties of the recombinant luciferase.

WOS
Держатели документа:
Russian Acad Sci, Shemyakin Ovchinnikov Inst Bioorgan Chem, Moscow, Russia.
Russian Acad Sci, Inst Biophys, Fed Res Ctr, Krasnoyarsk Sci Ctr,Siberian Branch, Krasnoyarsk, Russia.

Доп.точки доступа:
Gorokhovatsky, A. Yu; Chepurnykh, T., V; Shcheglov, A. S.; Mokrushina, Yu A.; Baranova, M. N.; Goncharuk, S. A.; Purtov, K., V; Petushkov, V. N.; Rodionova, N. S.; Yampolsky, I., V; Russian Science FoundationRussian Science Foundation (RSF) [16-14-00052-P]; President's grant for state support of the leading scientific schools of the Russian Federation [NSh-2605.2020.4]

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The production of highly active recombinant 16.5 kDa-isoform of Metridia longa luciferase, the smallest copepod luciferase [Text] / M. . Larionova [et al.] // Luminescence. - 2014. - Vol. 29. - P79-80. - Cited References: 3 . - ISSN 1522-7235. - ISSN 1522-7243
Рубрики:
EXPRESSION

WOS
Держатели документа:
[Larionova, Marina
Markova, Svetlana
Burakova, Ludmila
Vysotski, Eugene] Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk, Russia
[Larionova, Marina] Siberian Fed Univ, Inst Fundamental Biol & Biotecnol, Lab Bioluminescence Biotechnol, Krasnoyarsk, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Larionova, M...; Markova, S...; Burakova, L...; Vysotski, E...

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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 light-sensitive photoprotein berovin from the bioluminescent ctenophore Beroe abyssicola: a novel type of Ca2+-regulated photoprotein / S. V. Markova [et al.] // FEBS J. - 2012. - Vol. 279, Is. 5. - P856-870, DOI 10.1111/j.1742-4658.2012.08476.x. - Cited References: 63. - The authors thank Natalia Chervyakova from Department of Zoology of Invertebrates of Moscow State University for the photo of the White Sea ctenophore Beroe abyssicola. This work was supported by RFBR grant 09-04-00172, by grant 64987.2010.4, Molecular and Cellular Biology program of RAS, and Bayer Pharma AG (Germany). . - ISSN 1742-464X

РУБ Biochemistry & Molecular Biology
Рубрики:
CALCIUM-ACTIVATED PHOTOPROTEINS
   C-TERMINAL PROLINE
   SEQUENCE-ANALYSIS
   MNEMIOPSIS-SP
   COELENTERAZINE-BINDING
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   CRYSTAL-STRUCTURES
   EXCITED-STATE
   CDNA CLONING
Кл.слова (ненормированные):
bioluminescence -- calcium -- coelenterazine -- luciferase -- mammalian expression
Аннотация: Light-sensitive Ca2+-regulated photoproteins are responsible for the bright bioluminescence of ctenophores. Using functional screening, four full-size cDNA genes encoding the same 208-amino-acid polypeptide were isolated from two independent cDNA libraries prepared from two Beroe abyssicola specimens. Sequence analysis revealed three canonical EF-hand calcium-binding sites characteristic of Ca2+-regulated photoproteins, but a very low degree of sequence identity (2729%) with aequorin-type photoproteins, despite functional similarities. Recombinant berovin was expressed in Escherichia coli cells, purified, converted to active photoprotein and characterized. Active berovin has absorption maxima at 280 and 437 nm. The Ca2+-discharged protein loses visible absorption, but exhibits a new absorption maximum at 335 nm. The berovin bioluminescence is blue (?max = 491 nm) and a change in pH over the range 6.09.5 has no significant effect on the light emission spectrum. By contrast, the fluorescence of Ca2+-discharged protein (?ex = 350 nm) is pH sensitive: at neutral pH the maximum is at 420 nm and at alkaline pH there are two maxima at 410 and 485 nm. Like native ctenophore photoproteins, recombinant berovin is also inactivated by light. The Ca2+ concentrationeffect curve is a sigmoid with a slope on a loglog plot of similar to 2.5. Although this curve for berovin is very similar to those obtained for obelin and aequorin, there are evident distinctions: berovin responds to calcium changes at lower concentrations than jellyfish photoproteins and its Ca2+-independent luminescence is low. Recombinant berovin was successfully expressed in mammalian cells, thereby demonstrating potential for monitoring intracellular calcium.

Держатели документа:
[Vysotski, Eugene S.] Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia
[Markova, Svetlana V.
Burakova, Ludmila P.
Malikova, Natalia P.
Frank, Ludmila A.
Vysotski, Eugene S.] Siberian Fed Univ, Dept Biophys, Krasnoyarsk, Russia
[Golz, Stefan] Bayer Pharma AG, Global Drug Discovery, Wuppertal, Germany
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

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

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10.

The intrinsic fluorescence of apo-obelin and apo-aequorin and use of its quenching to characterize coelenterazine binding [Text] / E. V. Eremeeva [et al.] // FEBS Lett. - 2009. - Vol. 583, Is. 12. - P1939-1944, DOI 10.1016/j.febslet.2009.04.043. - Cited References: 28. - We thank Prof. John Lee for valuable suggestions and providing constructive criticisms. The work was supported by Wageningen University Sandwich PhD-Fellowship Program, Grants 02.512.12. 2006 and 1211.2008.4 of Ministry of Education and Science of Russian Federation, MCB Program of RAS, and by Grant No. 2 of SB RAS. . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
CRYSTAL-STRUCTURE
   CA2+-REGULATED PHOTOPROTEINS
   VIOLET BIOLUMINESCENCE
   ANGSTROM RESOLUTION
   RECOMBINANT OBELIN
   W92F OBELIN
   CALCIUM
   REGENERATION
   APOAEQUORIN
   EXPRESSION
Кл.слова (ненормированные):
Bioluminescence -- Photoprotein -- Trp fluorescence
Аннотация: The intrinsic fluorescence of two apo-photoproteins has been characterized and its concentration-dependent quenching by coelenterazine has been for the first time applied to determine the apparent dissociation constants for coelenterazine binding with apo-aequorin (1.2 +/- 0.12 mu M) and apo-obelin (0.2 +/- 0.04 mu M). Stopped-flow measurements of fluorescence quenching showed that coelenterazine binding is a millisecond-scale process, in contrast to the formation of an active photoprotein complex taking several hours. This finding evidently shows that the rate-limiting step of active photoprotein formation is the conversion of coelenterazine into its 2-hydroperoxy derivative. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Держатели документа:
[Eremeeva, Elena V.
Markova, Svetlana V.
Vysotski, Eugene S.] Russian Acad Sci, Inst Biophys, Photobiol Lab, Siberian Branch, Krasnoyarsk 660036, Russia
[Eremeeva, Elena V.
Westphal, Adrie H.
Visser, Antonie J. W. G.
van Berkel, Willem J. H.] Wageningen Univ, Biochem Lab, NL-6703 HA Wageningen, Netherlands
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Eremeeva, E.V.; Markova, S.V.; Westphal, A.H.; Visser, AJWG; van Berkel, WJH; Vysotski, E.S.; Wageningen University Sandwich PhD-Fellowship Program [02.512.12. 2006]; Ministry of Education and Science of Russian Federation, MCB Program of RAS [1211.2008.4]; SB RAS

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11.

The Hybrid Protein ZZ-OL as an Analytical Tool for Biotechnology Research / V. V. Krasitskaya, E. E. Bashmakova, A. N. Kudryavtsev [et al.] // Russ. J. Bioorg. Chem. - 2020. - Vol. 46, Is. 6. - P1004-1010, DOI 10.1134/S106816202006014X. - Cited References:14. - The study was partially supported by a grant of the President of the Russian Federation for Young Scientists, the Candidates of Sciences (project MK-772.2020.4 in the part involving the synthesis and analysis of variants of proteins with melanoma-inhibiting activity) and a grant of the Russian Science Foundation (project no. 16-14-10296 in the part involving the bioluminescence analysis of binding of DNA aptamers to targets). . - ISSN 1068-1620. - ISSN 1608-330X

РУБ Biochemistry & Molecular Biology + Chemistry, Organic

Кл.слова (ненормированные):
С -- а -- (2+)-regulated photoprotein obelin -- proZZ -- hybrid -- protein -- IgG -- bioluminescence assay
Аннотация: The gene of the hybrid protein that encodes the double synthetic fragment proZZ of the immunoglobulin-binding domain of protein A of Staphylococcus aureus and apo-obelin joined by a short linker has been cloned. The corresponding hybrid protein has been obtained by expression in Escherichia coli cells. The protein activated with a substrate (coelenterazine) possesses the bioluminescent Ca2+-dependent activity of the photoprotein close to that of recombinant wild-type obelin, and the immunoglobulin-binding ability of protein A. It has been shown that the hybrid can be used as a highly sensitive label to detect antibodies and estimate their affinity and interaction with recombinant proteins, as well as in investigations of other kinds.

WOS
Держатели документа:
Russian Acad Sci, Fed Res Ctr, Krasnoyarsk Res Ctr, Siberian Branch,Inst Biophys, Akad Gorodok 50-50, Krasnoyarsk 660036, Russia.
Russian Acad Sci, Inst Chem Biol & Fundamental Med, Siberian Branch, Novosibirsk 630090, Russia.

Доп.точки доступа:
Krasitskaya, V. V.; Bashmakova, E. E.; Kudryavtsev, A. N.; Vorobjeva, M. A.; Shatunova, E. A.; Frank, L. A.; Russian FederationRussian Federation [MK-772.2020.4]; Russian Science FoundationRussian Science Foundation (RSF) [16-14-10296]

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12.

THE ENTRY INTO S-PERIOD OF NUCLEI IN HETERODIKARYONS MODIFIED BY THE CYCLOHEXIMIDE [Текст] / N. A. SETKOV, V. N. KAZAKOV, T. V. ANDREEVA // TSITOLOGIYA. - 1991. - Vol. 33, Is. 12. - P. 73-78. - Cited References: 16 . - ISSN 0041-3771

РУБ Cell Biology
Рубрики:
NIH 3T3 CELLS
   DNA-SYNTHESIS
   RESTING CELLS
   C-MYC
   FUSION
   FIBROBLASTS
   EXPRESSION
   GENES
Аннотация: Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts were fused with serum-stimulated (10%) proliferating cells to elucidate mechanisms of entering into S-period operating in the nuclei of the heterokaryons under the effect of cycloheximide - an inhibitor of protein synthesis. Using radioautography DNA synthesis was investigated in mono-, homo- and heterodikaryons. After short (0.5-3.0 h) depressing of protein synthesis, the nuclei of stimulated cells in heterokaryons were found to enter into S-period. Under these conditions no induction of DNA synthesis was found in the nuclei of resting cells in heterodikaryons. In other experiments, resting cells were under the effect of cycloheximide during 2-4 h before the fusion, that led to a great induction of DNA synthesis in the nuclei of these cells in heterodikaryons. The data obtained are consistent with the idea of fibroblast transition to the rest under the action of labile proteins-repressors.

WOS : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
SETKOV, N.A.; KAZAKOV, V.N.; ANDREEVA, T.V.

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13.

The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells / S. V. Markova [et al.] // J. Photochem. Photobiol. B-Biol. - 2017. - Vol. 175. - P51-57, DOI 10.1016/j.jphotobiol.2017.08.024. - Cited References:30. - These studies were funded by RFBR and the Government of Krasnoyarsk Territory according to the research project No. 16-44-242099 and the state budget allocated to the fundamental research at the Russian Academy of Sciences (project No. 0356-2016-0712). . - ISSN 1011-1344

РУБ Biochemistry & Molecular Biology + Biophysics
Рубрики:
GAUSSIA-PRINCEPS LUCIFERASE
   ESCHERICHIA-COLI
   EXPRESSION
   PROTEIN
Кл.слова (ненормированные):
Copepod luciferase -- Disulfide bonds -- Cysteine-rich protein -- Oxidative -- refolding
Аннотация: The bioluminescence of a marine copepod Metridia Tonga is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (lambda(max) = 480 nm). 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 S-S intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. colt cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6 mg/L, the application of E. coil cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies.

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

Доп.точки доступа:
Markova, Svetlana V.; Larionova, Marina D.; Gorbunova, Darya A.; Vysotski, Eugene S.; RFBR; Government of Krasnoyarsk Territory [16-44-242099]; Russian Academy of Sciences [0356-2016-0712]

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14.

    The C-terminal tyrosine deletion in mitrocomin increases its bioluminescent activity [Text] / L. . Burakova [et al.] // Luminescence. - 2014. - Vol. 29. - P84-84. - Cited References: 6 . - ISSN 1522-7235. - ISSN 1522-7243
Рубрики:
PHOTOPROTEIN
   EXPRESSION
   AEQUORIN
   CLONING
   CDNA

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Держатели документа:
[Burakova, Liudmila
Natashin, Pavel
Markova, Svetlana
Eremeeva, Elena
Vysotsky, Eugene] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk, Russia
[Burakova, Liudmila
Natashin, Pavel
Markova, Svetlana
Eremeeva, Elena
Vysotsky, Eugene] Siberian Fed Univ, Krasnoyarsk, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Burakova, L...; Natashin, P...; Markova, S...; Eremeeva, E...; Vysotsky, E...

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15.

Study of the immunogenicity of the VP2 protein of canine parvovirus produced using an improved Baculovirus expression system / D. Chang, Y. Liu, Y. Chen [et al.] // BMC Vet. Res. - 2020. - Vol. 16, Is. 1. - Ст. 202, DOI 10.1186/s12917-020-02422-3 . - ISSN 1746-6148
Кл.слова (ненормированные):
Baculovirus expression system -- Canine parvovirus -- VP2 protein -- canine parvovirus vaccine -- protein VP2 -- recombinant protein -- unclassified drug -- virus antibody -- virus vaccine -- affinity chromatography -- animal experiment -- antibody titer -- Article -- baculovirus expression system -- Canine parvovirus -- controlled study -- DNA transposition -- enzyme linked immunosorbent assay -- female -- fluorescence microscopy -- gene expression level -- hemagglutination inhibition -- hemagglutination inhibition test -- immunogenicity -- mouse -- nonhuman -- parvovirus infection -- protein expression -- Sf9 cell line -- vaccination -- Western blotting
Аннотация: Background: Canine parvovirus (CPV) is now recognized as a serious threat to the dog breeding industry worldwide. Currently used CPV vaccines all have their specific drawbacks, prompting a search for alternative safe and effective vaccination strategies such as subunit vaccine. VP2 protein is the major antigen targeted for developing CPV subunit vaccine, however, its production in baculovirus expression system remains challenging due to the insufficient yield. Therefore, our study aims to increase the VP2 protein production by using an improved baculovirus expression system and to evaluate the immunogenicity of the purified VP2 protein in mice. Results: The results showed that high-level expression of the full length VP2 protein was achieved using our modified baculovirus expression system. The recombinant virus carrying two copies of VP2 gene showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein reacted with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody. BALB/c mice were intramuscularly immunized with purified VP2 protein twice at 2 week intervals. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. Conclusions: Full length CPV VP2 protein was expressed at high level and purified efficiently. Moreover, it stimulated mice to produce high level of antibodies with hemmaglutination inhibition properties. The VP2 protein expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection. © 2020 The Author(s).

Scopus
Держатели документа:
Henan Provincal Engineering and Technology Center of Health Products for Livestock and Poultry, Key Laboratory of Ecological Security, Collab. Innov. Ctr. of Water Secty. for Water Src. Reg. of Mid-line of S.-to-N. Diversion Proj. of Henan Prov., School of Agricultural Engineering, Nanyang Normal University, Nanyang, 473061, China
Institute of Biophysics, Siberian Branch, Russian Academy of Science, Federal Research Center Krasnoyarsk Science Center SB RAS, Krasnoyarsk, 660036, Russian Federation

Доп.точки доступа:
Chang, D.; Liu, Y.; Chen, Y.; Hu, X.; Burov, A.; Puzyr, A.; Bondar, V.; Yao, L.

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16.

Study of the immunogenicity of the VP2 protein of canine parvovirus produced using an improved Baculovirus expression system / D. Chang, Y. K. Liu, Y. Y. Chen [et al.] // BMC Vet. Res. - 2020. - Vol. 16, Is. 1. - Ст. 202, DOI 10.1186/s12917-020-02422-3. - Cited References:30. - This work was financially supported by the National Natural Science Foundation of China (No. 31870917), The program for Innovative Research Team of Science and Technology in University of Henan Province (No. 20IRTSTHN024) and Key Scientific Research Projects of Colleges and Universities in Henan Province of China (No. 18B230008). The funding bodies played no role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript. . - ISSN 1746-6148

РУБ Veterinary Sciences
Рубрики:
VIRUS-LIKE PARTICLES
   ESCHERICHIA-COLI
   GENETIC-ANALYSIS
   CPV-VP2
Кл.слова (ненормированные):
Canine parvovirus -- VP2 protein -- Baculovirus expression system
Аннотация: Background Canine parvovirus (CPV) is now recognized as a serious threat to the dog breeding industry worldwide. Currently used CPV vaccines all have their specific drawbacks, prompting a search for alternative safe and effective vaccination strategies such as subunit vaccine. VP2 protein is the major antigen targeted for developing CPV subunit vaccine, however, its production in baculovirus expression system remains challenging due to the insufficient yield. Therefore, our study aims to increase the VP2 protein production by using an improved baculovirus expression system and to evaluate the immunogenicity of the purified VP2 protein in mice. Results The results showed that high-level expression of the full length VP2 protein was achieved using our modified baculovirus expression system. The recombinant virus carrying two copies of VP2 gene showed the highest expression level, with a productivity of 186 mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein reacted with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody. BALB/c mice were intramuscularly immunized with purified VP2 protein twice at 2 week intervals. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. Conclusions Full length CPV VP2 protein was expressed at high level and purified efficiently. Moreover, it stimulated mice to produce high level of antibodies with hemmaglutination inhibition properties. The VP2 protein expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.

WOS
Держатели документа:
Nanyang Normal Univ, Sch Agr Engn, Henan Provincal Engn & Technol Ctr Hlth Prod Live, Nanyang 473061, Peoples R China.
Nanyang Normal Univ, Sch Agr Engn, Key Lab Ecol Secur, Nanyang 473061, Peoples R China.
Nanyang Normal Univ, Sch Agr Engn, Collaborat Innovat Ctr Water Secur Water Source R, Nanyang 473061, Peoples R China.
Russian Acad Sci, Fed Res Ctr, Krasnoyarsk Sci Ctr, Inst Biophys,Siberian Branch, Krasnoyarsk 660036, Russia.

Доп.точки доступа:
Chang, Dao; Liu, Yangkun; Chen, Yangyang; Hu, Xiaomin; Burov, Andrey; Puzyr, Alexey; Bondar, Vladimir; Yao, Lunguang; National Natural Science Foundation of ChinaNational Natural Science Foundation of China [31870917]; program for Innovative Research Team of Science and Technology in University of Henan Province [20IRTSTHN024]; Key Scientific Research Projects of Colleges and Universities in Henan Province of China [18B230008]

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17.

Stability of recombinant plasmids in transgenic microorganisms under different environmental conditions / L. Yu. Popova [et al.] // Microbiology. - 2001. - Vol. 70, Is. 6. - P685-691 . - ISSN 0026-2617
Кл.слова (ненормированные):
Antibiotic resistance -- Bioluminescence -- Interferon -- Recombinant plasmids -- Transgenic microorganisms
Аннотация: The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms. В© 2001 MAIK "Nauka/Interperiodica".

Scopus
Держатели документа:
Institute of Biophysics, Siberian Division, Russian Academy of Sciences, Krasnoyarsk, 660036, Russian Federation : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Popova, L.Yu.; Maksimova, E.E.; Lobova, T.I.; Kargatova, T.V.; Boyandin, A.N.; Krylova, T.Yu.; Pechurkin, N.S.

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18.

Stability of recombinant plasmids in transgenic microorganisms under different environmental conditions [Text] / L. Y. Popova [et al.] // Microbiology. - 2001. - Vol. 70, Is. 6. - P. 685-691, DOI 10.1023/A:1013187815739. - Cited References: 15 . - ISSN 0026-2617

РУБ Microbiology
Рубрики:
BACTERIA
Кл.слова (ненормированные):
recombinant plasmids -- transgenic microorganisms -- antibiotic resistance -- bioluminescence -- interferon
Аннотация: The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms.

WOS
Держатели документа:
Russian Acad Sci, Siberian Div, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Popova, L.Y.; Maksimova, E.E.; Lobova, T.I.; Kargatova, T.V.; Boyandin, A.N.; Krylova, T.Y.; Pechurkin, N.S.

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19.

Stability of recombinant plasmids in transgenic microorganisms under different environmental conditions / L. Yu. Popova [и др.] // Mikrobiologiya. - 2001. - Vol. 70, Is. 6. - С. 796-803 . - ISSN 0026-3656
Кл.слова (ненормированные):
Antibiotic resistance -- Bioluminescence interferon -- Recombinant plasmids -- Transgenic microorganisms -- article -- Bacillus subtilis -- culture medium -- Escherichia coli -- genetic engineering -- genetic recombination -- genetics -- R factor -- Bacillus subtilis -- Culture Media -- Escherichia coli -- Genetic Engineering -- R Factors -- Recombination, Genetic
Аннотация: The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms.

Scopus
Держатели документа:
Institute of Biophysics, Siberian Division, Russian Academy of Sciences, Krasnoyarsk, 660036, Russian Federation : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Popova, L.Yu.; Maksimova, E.E.; Lobova, T.I.; Kargatova, T.V.; Boyandin, A.N.; Krylova, T.Yu.; Pechurkin, N.S.

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20.

Spectral tuning of obelin bioluminescence by mutations of Trp92 [Text] / N. P. Malikova [et al.] // FEBS Lett. - 2003. - Vol. 554, Is. 01.02.2013. - P184-188, DOI 10.1016/S0014-5793(03)01166-9. - Cited References: 13 . - ISSN 0014-5793

РУБ Biochemistry & Molecular Biology + Biophysics + Cell Biology
Рубрики:
VIOLET BIOLUMINESCENCE
   W92F OBELIN
   AEQUORIN
   PURIFICATION
   EXPRESSION
   EMISSION
   CLONING
Кл.слова (ненормированные):
photoprotein -- aequorin -- calcium -- fluorescence
Аннотация: The Ca2+-regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence (lambda(max)=390 nm) and monomodal fluorescence (lambda(max)=425 nm) of the product. Results are interpreted by an excited state proton transfer mechanism involving the substituent side group and His22 in the binding cavity. (C) 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Держатели документа:
Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
Russian Acad Sci, Siberian Branch, Photobiol Lab, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

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

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