Rabuesak KhumthongChanan AngsuthanasombatSakol PanyimGerd KatzenmeierMahidol University2018-07-242018-07-242002-03-01Journal of Biochemistry and Molecular Biology. Vol.35, No.2 (2002), 206-21212258687122586872-s2.0-0036004555https://repository.li.mahidol.ac.th/handle/20.500.14594/20077The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3( pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates. © BSRK & Springer-Verlag 2002.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS