Linked Life Data

10744671

Source:http://linkedlifedata.com/resource/pubmed/id/10744671

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
14
pubmed:dateCreated
2000-5-8
pubmed:abstractText
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 coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) 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 Gln 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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
7
pubmed:volume
275
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9963-9
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:10744671-Amino Acid Sequence, pubmed-meshheading:10744671-Amino Acids, Diamino, pubmed-meshheading:10744671-Chromatography, Affinity, pubmed-meshheading:10744671-Chromatography, Gel, pubmed-meshheading:10744671-Cloning, Molecular, pubmed-meshheading:10744671-Coenzymes, pubmed-meshheading:10744671-Conserved Sequence, pubmed-meshheading:10744671-Dengue Virus, pubmed-meshheading:10744671-Endopeptidases, pubmed-meshheading:10744671-Enzyme Activation, pubmed-meshheading:10744671-Escherichia coli, pubmed-meshheading:10744671-Kinetics, pubmed-meshheading:10744671-Protein Precursors, pubmed-meshheading:10744671-RNA Helicases, pubmed-meshheading:10744671-Recombinant Fusion Proteins, pubmed-meshheading:10744671-Serine Endopeptidases, pubmed-meshheading:10744671-Substrate Specificity, pubmed-meshheading:10744671-Viral Nonstructural Proteins
pubmed:year
2000
pubmed:articleTitle
Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't