Publication: The Two-Component Protease NS2B-NS3 of Dengue Virus Type 2: Cloning, Expression in Escherichia coli and Purification of the NS2B, NS3(pro) and NS2B-NS3 Proteins
5
Issued Date
2000-07-31
Resource Type
ISSN
12258687
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2-s2.0-0034371094
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Biochemistry and Molecular Biology. Vol.33, No.4 (2000), 294-299
Suggested Citation
Veerawat Champreda, Rabuesak Khumthong, Benchamas Subsin, Chanan Angsuthanasombat, Sakol Panyim, Gerd Katzenmeier The Two-Component Protease NS2B-NS3 of Dengue Virus Type 2: Cloning, Expression in Escherichia coli and Purification of the NS2B, NS3(pro) and NS2B-NS3 Proteins. Journal of Biochemistry and Molecular Biology. Vol.33, No.4 (2000), 294-299. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/25861
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Title
The Two-Component Protease NS2B-NS3 of Dengue Virus Type 2: Cloning, Expression in Escherichia coli and Purification of the NS2B, NS3(pro) and NS2B-NS3 Proteins
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Abstract
Proteolytic processing of the dengue virus serotype 2 polyprotein precursor is catalyzed by a host signal peptidase and a virus encoded two-component protease consisting of the nonstructural proteins, NS2B and NS3. We expressed in Escherichia coli the NS2B, NS3(pro) and NS2B-NS3 proteins from the dengue virus type 2 strain 16681 as N-terminal fusions with a hexahistidine affinity tag under the control of the inducible trc promoter. All fusion proteins were purified to >90% purity by detergent extraction of inclusion bodies and a single step metal chelate chromatography. Proteins were refolded on-column and recovered with yields of 0.5, 6.0 and 1.0 mg/l of E. coli culture that was grown to OD600 = 1.0 for NS2B, NS3(pro) and NS2B-NS3, respectively. Purified proteins gave strong signals in Western blots using Ni2+-nitrilotriacetic add as a probe for the presence of the polyHis tag. During the purification process, (His)6NS2B-NS3 was apparently not autoproteolytically cleaved at the NS2B/NS3 site.