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1.

001-es BibID:BIBFORM008380
Első szerző:Bagossi Péter (biokémikus, vegyész)
Cím:Discovery and significance of new Human T-lymphotropic viruses : HTVL-3 and HTLV-4 / Bagossi, P., Bander, P., Bozóki, B., Tőzsér, J.
Dátum:2009
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Expert Review of Anti-Infective Therapy. - 7 : 10 (2009), p. 1235-1249. -
További szerzők:Bander Pálma (1979-) (molekuláris biológus) Bozóki Beáta (1986-) (molekuláris biológus) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Internet cím:elektronikus változat
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2.

001-es BibID:BIBFORM088520
035-os BibID:(cikkazonosító)E7686 (scopus)85092599352 (wos)000583012300001
Első szerző:Bozóki Beáta (molekuláris biológus)
Cím:Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates / Bozóki Beáta, Mótyán János András, Hoffka Gyula, Waugh David S., Tőzsér József
Dátum:2020
ISSN:1661-6596 1422-0067
Megjegyzések:The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1·, and P2· variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1· amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
VEEV
Venezuelan equine encephalitis virus
nsp2
protease
alphavirus
alphaviral protease
non-structural protein
group IV virus
specificity
Megjelenés:International Journal Of Molecular Sciences. - 21 : 20 (2020), p. 1-26. -
További szerzők:Mótyán János András (1981-) (biokémikus, molekuláris biológus) Hoffka Gyula (1992-) (vegyész) Waugh, David S. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:NKFI-125238
Egyéb
GINOP-2.3.2-15-2016-00044
GINOP
TÁMOP 4.2.4B/2-11/1-2012-0001
Egyéb
NKFIH-1150-6/2019
Egyéb
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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3.

001-es BibID:BIBFORM076746
Első szerző:Bozóki Beáta (molekuláris biológus)
Cím:Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation / Bozóki Beáta, Mótyán János András, Miczi Márió, Gazda Lívia Diána, Tőzsér József
Dátum:2019
ISSN:1940-087X
Megjegyzések:Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes. In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
recombinant fusion protein substrate
protease assay
Ni-NTA magnetic agarose beads
fluorescent protein
in-gel renaturation
human immunodeficiency virus type 1 protease
Megjelenés:Journal of Visualized Experiments. - 143 (2019), p. 1-15. -
További szerzők:Mótyán János András (1981-) (biokémikus, molekuláris biológus) Miczi Márió Gazda Lívia Diána (1989-) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:GINOP-2.3.2-15-2016-00044
GINOP
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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4.

001-es BibID:BIBFORM072540
Első szerző:Bozóki Beáta (molekuláris biológus)
Cím:A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening / Beáta Bozóki, Lívia Gazda, Ferenc Tóth, Márió Miczi, János András Mótyán, József Tőzsér
Dátum:2018
ISSN:0003-2697
Megjegyzések:In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Analytical Biochemistry. - 540-541 (2018), p. 52-63. -
További szerzők:Gazda Lívia Diána (1989-) Tóth Ferenc (1980-) (molekuláris biológus) Miczi Márió Mótyán János András (1981-) (biokémikus, molekuláris biológus) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:GINOP-2.3.2-15-2016-00044
GINOP
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5.

001-es BibID:BIBFORM095389
Első szerző:Miczi Márió
Cím:Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model / Márió Miczi, Ádám Diós, Beáta Bozóki, József Tőzsér, János András Mótyán
Dátum:2021
ISSN:1999-4915 1999-4915
Megjegyzések:first_page settings Open AccessArticle Development of a Bio-Layer Interferometry-Based Protease Assay Using HIV-1 Protease as a Model by Márió Miczi 1,2, Ádám Diós 2,3, Beáta Bozóki 1, József Tőzsér 1 [OrcID] and János András Mótyán 1,* [OrcID] 1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary 2 Doctoral School of Molecular Cell and Immune Biology, University of Debrecen, 4032 Debrecen, Hungary 3 Department of Pediatrics, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary * Author to whom correspondence should be addressed. Academic Editor: Alan Rein Viruses 2021, 13(6), 1183; https://doi.org/10.3390/v13061183 (registering DOI) Received: 30 April 2021 / Revised: 9 June 2021 / Accepted: 19 June 2021 / Published: 21 June 2021 (This article belongs to the Special Issue In Memory of Stephen Oroszlan) Download PDF Browse Figures Citation Export Abstract Proteolytic enzymes have great significance in medicine and the pharmaceutical industry and are applied in multiple fields of life sciences. Therefore, cost-efficient, reliable and sensitive real-time monitoring methods are highly desirable to measure protease activity. In this paper, we describe the development of a new experimental approach for investigation of proteolytic enzymes. The method was designed by the combination of recombinant fusion protein substrates and bio-layer interferometry (BLI). The protease (PR) of human immunodeficiency virus type 1 (HIV-1) was applied as model enzyme to set up and test the method. The principle of the assay is that the recombinant protein substrates immobilized to the surface of biosensor are specifically cleaved by the PR, and the substrate processing can be followed by measuring change in the layer thickness by optical measurement. We successfully used this method to detect the HIV-1 PR activity in real time, and the initial rate of the signal decrease was found to be proportional to the enzyme activity. Substrates representing wild-type and modified cleavage sites were designed to study HIV-1 PR's specificity, and the BLI-based measurements showed differential cleavage efficiency of the substrates, which was proven by enzyme kinetic measurements. We applied this BLI-based assay to experimentally confirm the existence of extended binding sites at the surface of HIV-1 PR. We found the measurements may be performed using lysates of cells expressing the fusion protein, without primary purification of the substrate. The designed BLI-based protease assay is high-throughput-compatible and enables real-time and small-volume measurements, thus providing a new and versatile approach to study proteolytic enzymes.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
protease assay
protease
HIV-1
BLItz
human immunodeficiency virus
bio-layer interferometry
recombinant fluorescent protein substrate
specificity
substrate specificity
recombinant protein
Megjelenés:Viruses-Basel. - 13 : 6 (2021), p. 1-20. -
További szerzők:Diós Ádám (1994-) (molekuláris biológus) Bozóki Beáta (1986-) (molekuláris biológus) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Mótyán János András (1981-) (biokémikus, molekuláris biológus)
Pályázati támogatás:TKP2020-IKA-04
Egyéb
EFOP-3.6.3-VEKOP-16-2017-00009
EFOP
GINOP-2.3.2-15-2016-00044
GINOP
GINOP-2.3.2-15-2016-00015
GINOP
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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6.

001-es BibID:BIBFORM072696
Első szerző:Mótyán János András (biokémikus, molekuláris biológus)
Cím:Data supporting Ni-NTA magnetic bead-based fluorescent protease assay using recombinant fusion protein substrates / Mótyán János András, Miczi Márió, Bozóki Beáta, Tőzsér József
Dátum:2018
ISSN:2352-3409
Megjegyzések:Data provided here are related to the research article entitled as ♭A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening'. Here we describe data related to the investigation of the properties of the His6-MBP-VSQNY?PIVQ-mApple recombinant protein substrate and its interactions with Ni-NTA magnetic beads, including the dependence of substrate attachment on incubation time and concentration. Data on the folding efficiency and conformational stability of the recombinant substrate assessed by tryptic digestion are also presented. We describe here the changes of fluorescent properties and binding abilities upon treatments commonly used for stopping enzymatic reactions: trichloroacetic acid (TCA) or heat treatment.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
recombinant fusion protein substrate
protease assay
fluorescent protein
Megjelenés:Data in Brief. - 18 (2018), p. 203-208. -
További szerzők:Miczi Márió Bozóki Beáta (1986-) (molekuláris biológus) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:GINOP-2.3.2-15-2016-00044
GINOP
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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