title: Fluorometric CCHFV OTU protease assay with potent inhibitors.
abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay.
authors.affiliation: Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, 34755, Turkey. fatih.kocabas@yeditepe.edu.tr.|Department of Education, North American University, Houston, TX, 77038, USA. fatih.kocabas@yeditepe.edu.tr.
authors.forename: Fatih
authors.initials: F
authors.lastname: Kocabas
authors.affiliation: Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, 34755, Turkey.
authors.forename: Galip S
authors.initials: GS
authors.lastname: Aslan
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005470:Fluorometry / Q000379:methods*; D003225:Hemorrhagic Fever Virus, Crimean-Congo / Q000201:enzymology* / Q000235:genetics; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000032:analysis*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; D010447:Peptide Hydrolases
keywords: Antivirals; CCHF; CCHFV; Crimean-Congo hemorrhagic fever virus; Expression and purification; Fluorescent assay; Viral OTU protease