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Steady-State Cleavage Kinetics for Dengue Virus Type 2 Ns2b-Ns3(Pro) Serine Protease With Synthetic Peptides

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Abstract:



The 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.





Keywords: assay; cleavage kinetics; dengue virus; kinetic; ns2b-ns3; peptide; serine protease; substrate

Document Type: Review Article

DOI: http://dx.doi.org/10.2174/0929866033408228

Publication date: February 1, 2003

More about this publication?
  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
ben/ppl/2003/00000010/00000001/art00003
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