Linked Life Data

14579737

Source:http://linkedlifedata.com/resource/pubmed/id/14579737

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2003-10-28
pubmed:abstractText
The success of recombinant protein expression/purification in Escherichia coli depends on a high-fidelity system rendering purified proteins free of confounding contaminants such as endotoxin. Here we report on the expression and purification of a cryptic plasminogen-derived domain, kringle 5, which was previously reported to specifically inhibit endothelial cell growth and, therefore, angiogenesis. Using a histidine (HIS)-tag expression and Ni(+)-NTA agarose purification system identical to previous reports, we found that our purified recombinant kringle 5 did inhibit endothelial cell growth, but this activity could not be eradicated by heat denaturing or proteolysis of kringle 5 with various proteases. This led us to suspect the presence of a contaminant in the purified samples. Quantitative endotoxin testing using a limulus amoebocyte lysate assay revealed that all samples purified by Ni(+)-NTA agarose alone harbored high concentrations of endotoxin that could not be removed by additional purification on anion exchange chromatography. Finally, when kringle 5 was rendered endotoxin-free by purification on reverse phase high-performance liquid chromatography (HPLC), there was a complete loss of endothelial cell growth inhibitory activity. These results strongly suggest that endotoxin-free recombinant kringle 5 may not possess anti-angiogenic activity and demonstrates that, especially in angiogenesis type assays, endotoxin contamination can lead to a misinterpretation of results.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0736-6205
pubmed:author
pubmed:issnType
Print
pubmed:volume
35
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
724-6, 728, 730 passim
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2003
pubmed:articleTitle
Removal of endotoxin by reverse phase HPLC abolishes anti-endothelial cell activity of bacterially expressed plasminogen kringle 5.
pubmed:affiliation
Department of Medicine, St. Vincent's Hospital, University of Melbourne, Corner of Princes and Regent Streets, Fitzroy, Melbourne, VIC, 3065, Australia. drew@medstv.unimelb.edu.au
pubmed:publicationType
Comparative Study, Evaluation Studies, Technical Report, Validation Studies