Linked Life Data

9882568

Source:http://linkedlifedata.com/resource/pubmed/id/9882568

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1999-2-22
pubmed:abstractText
Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli. The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E. coli as inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E. coli. In addition, the refolded CMV PR could be crystallized for X-ray diffraction.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
1046-5928
pubmed:author
pubmed:copyrightInfo
Copyright 1998 Academic Press.
pubmed:issnType
Print
pubmed:volume
14
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
343-52
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:9882568-Amino Acid Sequence, pubmed-meshheading:9882568-Bacteriophage lambda, pubmed-meshheading:9882568-Catalysis, pubmed-meshheading:9882568-Crystallization, pubmed-meshheading:9882568-Cytomegalovirus, pubmed-meshheading:9882568-Escherichia coli, pubmed-meshheading:9882568-Genetic Vectors, pubmed-meshheading:9882568-Histidine, pubmed-meshheading:9882568-Humans, pubmed-meshheading:9882568-Inclusion Bodies, pubmed-meshheading:9882568-Molecular Sequence Data, pubmed-meshheading:9882568-Peptides, pubmed-meshheading:9882568-Protein Folding, pubmed-meshheading:9882568-Recombinant Fusion Proteins, pubmed-meshheading:9882568-Sequence Alignment, pubmed-meshheading:9882568-Serine Endopeptidases, pubmed-meshheading:9882568-Solubility, pubmed-meshheading:9882568-Substrate Specificity
pubmed:year
1998
pubmed:articleTitle
Recombinant human cytomegalovirus protease with a C-terminal (His)6 extension: purification, autocatalytic release of the mature enzyme, and biochemical characterization.
pubmed:affiliation
Protein Science, Genomics, Bioprocess Research Preparations, Structural and Analytical and Medicinal Chemistry, Pharmacia & Upjohn, 301 Henrietta Street, Kalamazoo, Michigan, 49007, USA.
pubmed:publicationType
Journal Article, Comparative Study