Virus Genes

, Volume 51, Issue 2, pp 190–197 | Cite as

Fluorometric CCHFV OTU protease assay with potent inhibitors

Article

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.

Graphical Abstract

Keywords

Viral OTU protease Expression and purification Fluorescent assay Antivirals Crimean-Congo hemorrhagic fever virus CCHFV CCHF 

Supplementary material

11262_2015_1226_MOESM1_ESM.pdf (66 kb)
pET26b(+) vector was linearized with NdeI and NcoI enzymes. This was followed by In-Fusion cloning (Clontech) of linearized vector and two gene fragments (about 315-320 base pair long) with homology sequences. Supplementary material 1 (PDF 65 kb)
11262_2015_1226_MOESM2_ESM.pdf (121 kb)
Supplementary material 2 (PDF 121 kb)
11262_2015_1226_MOESM3_ESM.pdf (40 kb)
Supplementary material 3 (PDF 41 kb)

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Genetics and Bioengineering, Faculty of EngineeringYeditepe UniversityIstanbulTurkey
  2. 2.Department of EducationNorth American UniversityHoustonUSA

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