Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Beáta Bozóki, János András Mótyán, Márió Miczi, Lívia Diána Gazda, J. Tőzsér

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
JournalJournal of visualized experiments : JoVE
Issue number143
DOIs
Publication statusPublished - Jan 16 2019

Fingerprint

Recombinant Fusion Proteins
Assays
Peptide Hydrolases
Fusion reactions
Gels
Proteins
His-His-His-His-His-His
Viruses
Substrates
HIV-1
Protein Renaturation
Maltose-Binding Proteins
Fluorometry
Enzymes
Nickel
Mutagenesis
Sodium Dodecyl Sulfate
Sepharose
Tobacco
Maltose

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

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, J.

In: Journal of visualized experiments : JoVE, No. 143, 16.01.2019.

Research output: Contribution to journalArticle

Bozóki, Beáta ; Mótyán, János András ; Miczi, Márió ; Gazda, Lívia Diána ; Tőzsér, J. / Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation. In: Journal of visualized experiments : JoVE. 2019 ; No. 143.
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