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Fluorometric CCHFV OTU protease assay with potent inhibitors

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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.

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Acknowledgments

We thank Dr. Savas Kaya from Immunology Department, School of Medicine, Dicle University, Diyarbakir Turkey for his valuable input regarding MGLTools, and Autodock Vina. We thank Dr. Andrew J. Harvey from the Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, Turkey for his critical reading of the manuscript. We thank the support from Co-Funded Brain Circulation Scheme by The Scientific and Technological Research Council of Turkey (TÜBİTAK) and The Marie Curie Action COFUND of the 7th. Framework Programme (FP7) of the European Commission Grant#115C039, The Science Academy Young Scientist Award Program (BAGEP-2015, Turkey), funds provided by North American University, Houston, USA and Yeditepe University, Istanbul, Turkey.

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Authors and Affiliations

  1. Department of Genetics and Bioengineering, Faculty of Engineering, Yeditepe University, Istanbul, 34755, Turkey

    Fatih Kocabas & Galip S. Aslan

  2. Department of Education, North American University, Houston, TX, 77038, USA

    Fatih Kocabas

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  1. Fatih Kocabas
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  2. Galip S. Aslan
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Correspondence to Fatih Kocabas.

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Edited by Paul Schnitzler.

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11262_2015_1226_MOESM1_ESM.pdf

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)

Supplementary material 2 (PDF 121 kb)

Supplementary material 3 (PDF 41 kb)

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Kocabas, F., Aslan, G.S. Fluorometric CCHFV OTU protease assay with potent inhibitors. Virus Genes 51, 190–197 (2015). https://doi.org/10.1007/s11262-015-1226-5

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