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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
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
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