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

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 t...

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Main Authors: M. Junaid, C. Angsuthanasombat, J. E.S. Wikberg, N. Ali, G. Katzenmeier
Other Authors: University of Malakand
Format: Article
Published: 2018
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Online Access:https://repository.li.mahidol.ac.th/handle/123456789/31260
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spelling th-mahidol.312602018-10-19T11:37:35Z A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease M. Junaid C. Angsuthanasombat J. E.S. Wikberg N. Ali G. Katzenmeier University of Malakand Mahidol University Uppsala Universitet Khyber Medical University Biochemistry, Genetics and Molecular Biology 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, Km, kcat, and kcat/Kmfor the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Kmfor 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. © 2013 Pleiades Publishing, Ltd. 2018-10-19T04:37:35Z 2018-10-19T04:37:35Z 2013-08-01 Article Biochemistry (Moscow). Vol.78, No.8 (2013), 920-924 10.1134/S0006297913080099 16083040 00062979 2-s2.0-84882672686 https://repository.li.mahidol.ac.th/handle/123456789/31260 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84882672686&origin=inward
institution Mahidol University
building Mahidol University Library
continent Asia
country Thailand
Thailand
content_provider Mahidol University Library
collection Mahidol University Institutional Repository
topic Biochemistry, Genetics and Molecular Biology
spellingShingle Biochemistry, Genetics and Molecular Biology
M. Junaid
C. Angsuthanasombat
J. E.S. Wikberg
N. Ali
G. Katzenmeier
A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
description 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, Km, kcat, and kcat/Kmfor the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Kmfor 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. © 2013 Pleiades Publishing, Ltd.
author2 University of Malakand
author_facet University of Malakand
M. Junaid
C. Angsuthanasombat
J. E.S. Wikberg
N. Ali
G. Katzenmeier
format Article
author M. Junaid
C. Angsuthanasombat
J. E.S. Wikberg
N. Ali
G. Katzenmeier
author_sort M. Junaid
title A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
title_short A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
title_full A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
title_fullStr A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
title_full_unstemmed A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease
title_sort straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus ns2b(h)-ns3pro protease
publishDate 2018
url https://repository.li.mahidol.ac.th/handle/123456789/31260
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