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A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
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2013 (English)In: Biochemistry (Moscow), ISSN 0006-2979, E-ISSN 1608-3040, Vol. 78, no 8, 920-924 p.Article in journal (Refereed) Published
Abstract [en]

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to > 95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K (m), k (cat), and k (cat)/K (m) for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K (m), k (cat), and k (cat)/K (m) for Ac-nKRR-amc substrate were 100 mu M, 0.112 s(-1), and 1120 M-1 center dot s(-1), respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.

Place, publisher, year, edition, pages
2013. Vol. 78, no 8, 920-924 p.
Keyword [en]
Dengue virus, NS2B(H)-NS3pro protease, purification, assay, substrate
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-207635DOI: 10.1134/S0006297913080099ISI: 000323272100009OAI: oai:DiVA.org:uu-207635DiVA: diva2:649051
Available from: 2013-09-17 Created: 2013-09-17 Last updated: 2017-12-06Bibliographically approved

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Wikberg, Jarl E. S.

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