Linked Life Data

20709174

Source:http://linkedlifedata.com/resource/pubmed/id/20709174

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2011-1-28
pubmed:abstractText
DNA topoisomerase type II enzymes are well-validated targets of anti-bacterial and anti-cancer compounds. In order to facilitate discovery of these types of inhibitors human topoisomerase II in vitro assays can play an important role to support drug discovery processes. Typically, human topoisomerase II? proteins have been purified from human cell lines or as untagged proteins from yeast cells. This study reports a method for the rapid over-expression and purification of active GST-tagged human topoisomerase II? using the baculovirus mediated insect cell expression system. Expression of the GST fused protein was observed in the nuclear fraction of insect cells. High yields (40 mg/L i.e. 8 mg/10(9) cells) at >80% purity of this target was achieved by purification using a GST HiTrap column followed by size exclusion chromatography. Functional activity of GST-tagged human topoisomerase II? was demonstrated by ATP-dependent relaxation of supercoiled DNA in an agarose gel based assay. An 8-fold DNA-dependent increase in ATPase activity of this target compared to its intrinsic activity was also demonstrated in a high-throughput ATPase fluorescence based assay. Human topoisomerase II? inhibitors etoposide, quercetin and suramin were tested in the fluorescence assay. IC(50) values obtained were in good agreement with published data. These inhibitors also demonstrated ? 30-fold potency over the anti-bacterial topoisomerase II inhibitor ciprofloxacin in the assay. Collectively these data validated the enzyme and the high-throughput fluorescence assay as tools for inhibitor identification and selectivity studies.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1096-0279
pubmed:author
pubmed:copyrightInfo
Copyright © 2010. Published by Elsevier Inc.
pubmed:issnType
Electronic
pubmed:volume
76
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
165-72
pubmed:meshHeading
pubmed:year
2011
pubmed:articleTitle
High-yield production and characterization of biologically active GST-tagged human topoisomerase II? protein in insect cells for the development of a high-throughput assay.
pubmed:affiliation
Biological Reagents & Assay Development, GlaxoSmithKline R&D, New Frontiers Science Park, Harlow, Essex, UK. praveen.singh@kcl.ac.uk
pubmed:publicationType
Journal Article