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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
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
protease assay
human immunodeficiency virus
bio-layer interferometry
recombinant fluorescent protein substrate
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
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