Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

Yusof, R. and Clum, S. and Wetzel, M. and Murthy, H.M.K. and Padmanabhan, R. (2000) Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro. Journal of Biological Chemistry, 275 (14). pp. 9963-9969. ISSN 0021-9258

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Abstract

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia cole as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni2+ affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gin residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

Item Type: Article
Additional Information: Times Cited: 111 Yusof, R Clum, S Wetzel, M Murthy, HMK Padmanabhan, R
Subjects: R Medicine
Divisions: Faculty of Medicine
Depositing User: Ms Haslinda Lahuddin
Date Deposited: 11 Jul 2013 02:07
Last Modified: 11 Jul 2013 02:07
URI: http://eprints.um.edu.my/id/eprint/7148

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