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A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease

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Abstract

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 μM, 0.112 s−1, and 1120 M−1·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.

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Authors and Affiliations

  1. Department of Pharmacy, University of Malakand, Chakdara, 18550, Pakistan

    M. Junaid

  2. Laboratory of Molecular and Cellular Microbiology, Institute of Molecular Biosciences, Mahidol University, Salaya, 73170, Thailand

    M. Junaid, C. Angsuthanasombat & G. Katzenmeier

  3. Division of Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, 75124, Sweden

    J. E. S. Wikberg

  4. Department of Pharmacology, Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Khyber Pakhtunkhwa, 25000, Pakistan

    N. Ali

Authors
  1. M. Junaid
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  2. C. Angsuthanasombat
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  3. J. E. S. Wikberg
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  4. N. Ali
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  5. G. Katzenmeier
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Corresponding author

Correspondence to G. Katzenmeier.

Additional information

Published in Russian in Biokhimiya, 2013, Vol. 78, No. 8, pp. 1171–1177.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM13-043, July 14, 2013.

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Junaid, M., Angsuthanasombat, C., Wikberg, J.E.S. et al. A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease. Biochemistry Moscow 78, 920–924 (2013). https://doi.org/10.1134/S0006297913080099

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  • DOI: https://doi.org/10.1134/S0006297913080099

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