Rabuesak KhumthongPornwarat NiyomrattanakitSantad ChanprapaphChanan AngsuthanasombatSakol PanyimGerd KatzenmeierMahidol University2018-07-242018-07-242003-02-08Protein and Peptide Letters. Vol.10, No.1 (2003), 19-26092986652-s2.0-0037252890https://repository.li.mahidol.ac.th/handle/20.500.14594/20763The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for Km, kcat and kcat/Km were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.2174/0929866033408228