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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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Bioluminescent reporters for identification of gene allelic variants / V. V. Krasitskaya [et al.] // Russ. J. Bioorg. Chem. - 2012. - Vol. 38, Is. 3. - P298-305, DOI 10.1134/S1068162012030090. - Cited References: 13. - The authors thank the staff of Hematology Research Center (Krasnoyarsk Branch of Russian Academy of Medical Sciences) for providing DNA samples. The work was supported by the Integration Interdisciplinary Project of Siberian Branch of the Russian Academy of Sciences No. 76 and the Krasno yarsk Regional Fund for the support of scientific and technological activities. . - ISSN 1068-1620

РУБ Biochemistry & Molecular Biology + Chemistry, Organic
Рубрики:
COELENTERAZINE-BINDING PROTEIN
   RENILLA-MUELLERI
   LUCIFERASE
   PURIFICATION
   SUBSTRATE
   CLONING
   CDNA
Кл.слова (ненормированные):
SNP -- PEXT reaction -- obelin -- luciferase -- bioluminescent microassay
Аннотация: A method for single nucleotide polymorphism identification was developed, which was based on the primer extension reaction (PEXT) followed by bioluminescent solid-phase microassay. Recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent Renilla muelleri luciferase were used as reporters. The study was performed as an example of SNP genotyping of the human F5 gene encoding human Factor V Leiden polymorphism 1691 G -> A (R506Q). Genomic DNA was amplified by PCR using primers flanking polymorphic site of 140 base pairs. PCR products were used as templates for two PEXT reactions using two primers containing 3'-terminal nucleotides, which were complementary to either normal or mutant alleles. If the template and allele-specific primer were completely complementary, the latter was elongated with DNA polymerase. The resulting extension product contained biotin residue due to the presence of biotinylated deoxyuridine triphosphate (B-dUTP) in the reaction mixture. The products were analyzed using obelin-streptavidin conjugates. The optimal PEXT-reaction conditions were found, which ensured a high reliability of SNP genotyping. A new approach to simultaneously revealing both alleles in one well was developed using two bioluminescent reporters. The efficiency of the proposed approach was shown in the study of clinical DNA samples.

Держатели документа:
[Krasitskaya, V. V.
Burakova, L. P.
Frank, L. A.] Russian Acad Sci, Inst Biophys, Siberian Branch, Krasnoyarsk 660036, Akademgorodok, Russia
[Pyshnaya, I. A.] Russian Acad Sci, Siberian Branch, Inst Chem Biol & Fundamental Med, Novosibirsk 630090, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Krasitskaya, V.V.; Burakova, L.P.; Pyshnaya, I.A.; Frank, L.A.

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Ca2+-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay [Text] / V. V. Krasitskaya [et al.] // Anal. Bioanal. Chem. - 2011. - Vol. 401, Is. 8. - P2573-2579, DOI 10.1007/s00216-011-5343-2. - Cited References: 17. - The work was supported by a "Leading Scientific School" (N 64987.2010.4) grant from the President of the Russian Federation and the "Molecular and Cell Biology" Program from the RAS. . - ISSN 1618-2642

РУБ Biochemical Research Methods + Chemistry, Analytical
Рубрики:
BIOLUMINESCENT IMMUNOASSAY
   LUCIFERASE
   PURIFICATION
   RENIFORMIS
   MUELLERI
   OBELIN
   PHOTOPROTEIN
   EXPRESSION
   SUBSTRATE
   CLONING
Кл.слова (ненормированные):
Ca2+-triggered coelenterazine-binding protein (CBP) -- Renilla muelleri luciferase -- Bioluminescent solid-phase microassay
Аннотация: The recombinant Ca2+-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

Держатели документа:
[Korneeva, S. I.
Kudryavtsev, A. N.
Frank, L. A.] Siberian Fed Univ, Krasnoyarsk 660041, Russia
[Krasitskaya, V. V.
Markova, S. V.
Stepanyuk, G. A.
Frank, L. A.] Russian Acad Sci SB, Inst Biophys, Krasnoyarsk 660036, Russia
ИБФ СО РАН : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Krasitskaya, V.V.; Korneeva, S.I.; Kudryavtsev, A.N.; Markova, S.V.; Stepanyuk, G.A.; Frank, L.A.

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RNA aptamer against autoantibodies associated with multiple sclerosis and bioluminescent detection probe on its basis [] / M. A. Vorobjeva [et al.] // Anal. Chem. - 2014. - Vol. 86, Is. 5. - P2590-2594, DOI 10.1021/ac4037894 . - ISSN 0003-2700
Кл.слова (ненормированные):
Autoantibodies -- Detection probes -- Developed model -- High affinity -- Laboratory test -- Microplate assay -- Multiple sclerosis -- Myelin basic protein -- Antibodies -- RNA -- Bioluminescence
Аннотация: Nowadays, there are no specific laboratory tests for establishing the diagnosis of multiple sclerosis (MS). The presence of proteolytic autoantibodies against myelin basic protein is now considered as a characteristic feature of MS. New 2?-F-containing RNA aptamer of high affinity and specificity to these antibodies was selected. Covalent conjugate of this aptamer and Ca 2+-regulated photoprotein obelin was obtained for the first time and applied as a label in bioluminescent microplate assay to detect target antibodies. The developed model solid-phase microassay is simple, fast, and highly sensitive. © 2014 American Chemical Society.

Scopus
Держатели документа:
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk 630090, Russian Federation
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk 660036, Russian Federation
Siberian Federal University, Krasnoyarsk 660041, Russian Federation : 660036, Красноярск, Академгородок, д. 50, стр. 50

Доп.точки доступа:
Vorobjeva, M.A.; Krasitskaya, V.V.; Fokina, A.A.; Timoshenko, V.V.; Nevinsky, G.A.; Venyaminova, A.G.; Frank, L.A.

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Simultaneous Genotyping of Four Single Nucleotide Polymorphisms Associated with Risk Factors of Hemostasis Disorders [Text] / E. E. Bashmakova, V. V. Krasitskaya, L. A. Frank // Comb. Chem. High Throughput Screen. - 2015. - Vol. 18, Is. 10. - P930-936, DOI 10.2174/1386207318666150917095903. - Cited References:20. - The study was supported by the grant 14-14-01119 of the Russian Science Foundation. . - ISSN 1386-2073. - ISSN 1875-5402

РУБ Biochemical Research Methods + Chemistry, Applied + Pharmacology &
Рубрики:
ALLELE-SPECIFIC PCR
FACTOR-V-LEIDEN
BIOLUMINESCENT IMMUNOASSAY
Кл.слова (ненормированные):
SNP detection -- PEXT reaction -- photoprotein obelin -- bioluminescent -- microassay -- multiplex PCR
Аннотация: Multiplex simultaneous genotyping technique was developed for four polymorphisms in genes coding for blood coagulation factors and homocysteine metabolism which are considered as thrombophilia related mutations: FV Leiden, FII G20210A, MTHFR C677T, and FVII G10976A. It is based on primer extension reaction with the following bioluminescent solid-phase microassay. At that, two different in bioluminescence obelin mutants were applied to simultaneous detection of two gene allelic variants. The assay is carried out in microtiter plate format and provides fast and reliable genotyping of four single nucleotide polymorphisms in four different genes within 2.5 hours. A large number of clinical samples were analyzed and the obtained results were found to be in complete correlation with those obtained by using conventional RT-PCR techniques.

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Держатели документа:
Russian Acad Sci, Siberian Branch, Inst Biophys, Photobiol Lab, Krasnoyarsk 660036, Russia.
Siberian Fed Univ, Krasnoyarsk 660041, Russia.

Доп.точки доступа:
Bashmakova, Eugenia E.; Krasitskaya, Vasilisa V.; Frank, Ludmila A.; Russian Science Foundation [14-14-01119]

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Simultaneous genotyping of four single nucleotide polymorphisms associated with risk factors of hemostasis disorders / E. E. Bashmakova, V. V. Krasitskaya, L. A. Frank // Comb. Chem. High Throughput Screen. - 2015. - Vol. 18, Is. 10. - P930-936 . - ISSN 1386-2073
Кл.слова (ненормированные):
Bioluminescent microassay -- Multiplex PCR -- PEXT reaction -- Photoprotein obelin -- SNP detection
Аннотация: Multiplex simultaneous genotyping technique was developed for four polymorphisms in genes coding for blood coagulation factors and homocysteine metabolism which are considered as thrombophilia related mutations: FV Leiden, FII G20210A, MTHFR C677T, and FVII G10976A. It is based on primer extension reaction with the following bioluminescent solid-phase microassay. At that, two different in bioluminescence obelin mutants were applied to simultaneous detection of two gene allelic variants. The assay is carried out in microtiter plate format and provides fast and reliable genotyping of four single nucleotide polymorphisms in four different genes within 2.5 hours. A large number of clinical samples were analyzed and the obtained results were found to be in complete correlation with those obtained by using conventional RT-PCR techniques. © 2015 Bentham Science Publishers.

Scopus
Держатели документа:
Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, Russian Federation
Siberian Federal University, Svobodnii Ave., 79, Krasnoyarsk, Russian Federation

Доп.точки доступа:
Bashmakova, E. E.; Krasitskaya, V. V.; Frank, L. A.

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Bioluminescent aptamer-based solid-phase microassay to detect lung tumor cells in plasma / E. E. Bashmakova [et al.] // Talanta. - 2019. - Vol. 199. - P674-678, DOI 10.1016/j.talanta.2019.03.030. - Cited References:19. - The authors are grateful to the staff of Krasnoyarsk regional clinical oncology center named after A.I. Kryzhanovsky and particularly doctor Alexey V. Krat for the experimental material provided. This work was supported by the state budget allocated to the fundamental research at the Russian Academy of Sciences, project No. 0356-2017-0017. . - ISSN 0039-9140. - ISSN 1873-3573

РУБ Chemistry, Analytical
Рубрики:
CONJUGATED NANOPARTICLES
   CANCER
   COLLECTION
   LIGANDS
   PROBES
Кл.слова (ненормированные):
DNA aptamers -- Lung tumor -- Bioluminescent solid-phase microassay
Аннотация: Two high-affinity DNA aptamers for lung tumor cells were applied as biospecific elements in bioluminescent assay of patient blood. The oligonucleotide complementary to the 5' end of both aptamers carrying either biotin or Ca2+-regulated photoprotein obelin was used to form a sandwich-type analytical complex on the surfaces of magnetic streptavidin-activated microspherical particles. Clinical blood samples from cases of morphologically confirmed lung cancer and control samples were analyzed applying the developed assay. From the receiver operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers the sensitivity of 91.5% and the specificity of 75% (p < 0.001). The area under ROC curve with the value of 0.901 distinguishes well between the two groups under investigation.

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Держатели документа:
RAS, Krasnoyarsk Sci Ctr SB, Fed Res Ctr, Inst Biophys SB, Akademgorodok 50-50, Krasnoyarsk 660036, Russia.
Siberian Fed Univ, Svobodny Pr 79, Krasnoyarsk 660041, Russia.
State Med Univ, Partizana Zheleznyaka St 1, Krasnoyarsk 660022, Russia.

Доп.точки доступа:
Bashmakova, Eugenia E.; Krasitskaya, Vasilisa V.; Zamay, Galina S.; Zamay, Tatiana N.; Frank, Ludmila A.; Russian Academy of Sciences [0356-2017-0017]

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Bioluminescent aptamer-based sandwich-type assay of anti-myelin basic protein autoantibodies associated with multiple sclerosis / V. V. Krasitskaya [et al.] // Anal. Chim. Acta. - 2019. - Vol. 1064. - P112-118, DOI 10.1016/j.aca.2019.03.015. - Cited References:29. - This work was supported by the Russian Foundation for Basic Research (RFBR), Russia, under the grant No 17-315-50027; Russian State funded budget projects No. AAAA-A17-117013050026-4 and AAAA-A17-117020210021-7. . - ISSN 0003-2670. - ISSN 1873-4324

РУБ Chemistry, Analytical
Рубрики:
ANTIBODIES
BIOMARKERS
RNA
Кл.слова (ненормированные):
Bioluminescent microassay -- RNA aptamers -- Autoantibodies to myelin basic -- protein -- Multiple sclerosis
Аннотация: Bioluminescent solid-phase sandwich-type microassay was developed to detect multiple sclerosis (MS)-associated autoantibodies in human sera. The assay is based on two different 2'-F-Py RNA aptamers against the target autoantibodies as biospecific elements, and Ca2+-regulated photoprotein obelin as a reporter. The paper describes elaboration of the assay and its application to 91 serum samples from patients with clinically definite MS and 86 ones from individuals healthy in terms of MS. Based on the receiver-operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers sensitivity of 63.7% and specificity of 94.2%. The area under the ROC curve (AUC) value of 0.87 shows a good difference between the groups under investigation. The likelihood ratio of 10.97 proves the diagnostic value of the assay and its potential as one of the laboratory MS-tests. (C) 2019 Elsevier B.V. All rights reserved.

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Scopus
Держатели документа:
RAS, Krasnoyarsk Sci Ctr SB, Fed Res Ctr, Inst Biophys SB, Krasnoyarsk 660036, Russia.
RAS, Inst Chem Biol & Fundamental Med SB, Novosibirsk 630090, Russia.
State Med Univ, Krasnoyarsk 660022, Russia.

Доп.точки доступа:
Krasitskaya, Vasilisa V.; Chaukina, Valentina V.; Abroskina, Maria V.; Vorobyeva, Maria A.; Ilminskaya, Aleksandra A.; Kabilov, Marsel R.; Prokopenko, Semyon V.; Nevinsky, Georgy A.; Venyaminova, Alya G.; Frank, Ludmila A.; Russian Foundation for Basic Research (RFBR), Russia [17-315-50027]; Russian State [AAAA-A17-117013050026-4, AAAA-A17-117020210021-7]

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Development of the method to produce functionally active recombinant streptavidin in escherichia coli cells / E. E. Bashmakova, A. N. Kudryavtsev, L. A. Frank // J. Sib. Fed. Univ. - Biol. - 2020. - Vol. 13, Is. 2. - С. 218-229, DOI 10.17516/1997-1389-0324 . - ISSN 1997-1389
Кл.слова (ненормированные):
E. coli protein-producing strain -- Microanalysis -- Recombinant streptavidin
Аннотация: Streptavidin is a homotetrameric protein produced by Streptomyces avidinii, each subunit of which binds biotin (vitamin H), forming a stable complex (Kd = 10-15 M). Streptavidin-biotin coreaction is widely used in analytical systems, for targeted delivery of compounds, for affinity purification, etc. The aim of this study was to develop a rational technique to produce functionally active recombinant streptavidin. Recombinant Escherichia coli strains producing minimal core and full-sized streptavidin variants were obtained. The E. coli BL21 Codon Plus (DE3) RIPL, as host cells, and the pET19b plasmid carrying gene of minimally-sized core (miniSAV) or full-sized (SAV) streptavidin were used. Synthesis of miniSAV results in its localization as insoluble inclusion bodies. Denatured miniSAV yield was 130 mg per liter of E. coli c ulture. T he r enaturation g ives o nly 10- 15 % of the functionally active protein. Full-sized streptavidin localizes in the cytoplasm in a soluble state, but its toxicity causes low yield of the protein (10-13 mg per liter of the culture). The induction of SAV synthesis at the end of the logarithmic stage of cell growth was found to increase the yield of SAV approximately 2-fold. The yield of functionally active protein was 30 mg per liter culture. SAV was produced practically in individual state after affine chromatography on 2-iminobiotin agarose. One molecule of full-sized streptavidin bound 3.9 biotin molecules as was shown by colorimetric analysis using HABA (4-hydroxyazobenzene-2-carboxylic acid). Both streptavidins form sandwichtype complexes with biotinylated molecules in solid-phase microassay conditions. E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV strain was stable during storage with 20 % glycerol at -70 °C, which was shown by repeated two-year reseeding. The streptavidin producing strain (E. coli BL21 Codon Plus (DE3) RIPL/pET19bSAV) is deposited in the Collection for extremophile microorganisms and type cultures (Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk), No. 3505. The method for producing functionally active recombinant streptavidin developed in this study ensures its availability for biotechnological research. © Siberian Federal University. All rights reserved.

Scopus
Держатели документа:
Institute of Biophysics SB RAS, FRC Krasnoyarsk Science Center SB RAS, Krasnoyarsk, Russian Federation

Доп.точки доступа:
Bashmakova, E. E.; Kudryavtsev, A. N.; Frank, L. A.

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10.

A test system for tick-borne encephalitis virus detection based on bioluminescent immunoassay / A. N. Kudryavtsev, L. P. Burakova, K. A. Barinova, L. A. Frank // J. Sib. Fed. Univ. - Biol. - 2020. - Vol. 13, Is. 3. - С. 310-321, DOI 10.17516/1997-1389-0296 . - ISSN 1997-1389
Кл.слова (ненормированные):
Bioluminescent microassay -- Hybrid protein 14D5a-Rm7 -- Tick-borne encephalitis virus (TBEV)
Аннотация: The tick-borne encephalitis virus (TBEV) is the causative agent of one of the most severe human neuroinfections. The infection transmitted by ixodid ticks is spread throughout the forest and forest-steppe zones of the temperate climatic belt of the Eurasian continent, including the Siberian region of the Russian Federation. Despite the availability of commercial analytical systems for the detection of TBEV, the task of developing approaches to a quick and reliable analysis that can be performed routinely, particularly in environmental studies, remains topical. A solid-phase bioluminescent immunoassay for determining the tick-borne encephalitis virus (TBEV) in ticks was developed. The assay is based on the hybrid protein consisting of a modified thermostable version of Renilla muelleri luciferase and a single-chain mini-antibody to protein E. This unique protein had been obtained and investigated by the authors earlier. The current study describes the expression of the hybrid protein in two different strains of recombinant E. coli cells. The optimal conditions for obtaining a highly purified protein were found. The bioluminescent reaction of the luciferase domain was triggered with the help of the stable natural form of the substrate, a Ca-dependent coelenterazine-binding protein, the recombinant variant of which was obtained by the authors. The conditions for production and storage of the immunoassay components (the hybrid protein, the stable form of the luciferase substrate, and activated microplates) were determined. Using the developed test system, more than 900 tick samples were analyzed for TBEV. In terms of sensitivity (89.5%) and specificity (98.9%), the proposed method is not inferior to colorimetric detection and is much simpler and faster than the latter. © Siberian Federal University. All rights reserved.

Scopus
Держатели документа:
Institute of Biophysics SB RAS, FRC Krasnoyarsk Science Center SB RAS, Krasnoyarsk, Russian Federation
Siberian Federal University, Krasnoyarsk, Russian Federation

Доп.точки доступа:
Kudryavtsev, A. N.; Burakova, L. P.; Barinova, K. A.; Frank, L. A.

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