Linked Life Data

16125194

Source:http://linkedlifedata.com/resource/pubmed/id/16125194

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1-2
pubmed:dateCreated
2005-9-12
pubmed:abstractText
Production of diagnostic reagents in the yeast Pichia pastoris is particularly attractive since this organism is capable of expressing complex eukaryotic proteins in their correctly folded form and is amenable to large-scale fermentation at low cost. The potential of Schistosoma mansoni cathepsin B as a diagnostic antigen for human schistosomiasis has been previously established using both native and E. coli-derived recombinant proteins. However, when produced in P. pastoris we found that recombinant wild-type cathepsin B was preferentially secreted as a heterogeneously glycosylated molecule that migrated at 39 kDa, 41 kDa and a smear of >50 kDa on SDS-PAGE, and was susceptible to treatment with Endo H and PGNase F. The addition of yeast sugars to the cathepsin B caused it to react with IgG in the serum of both normal (non-infected) and schistosome-infected humans in immunoblotting and enzyme linked immunosorbent assays (ELISA). To avoid this non-specific reactivity, a non-glycosylated mutant form of cathepsin B, engineered by disrupting its potential glycosylation site, was produced. The non-glycosylated recombinant cathepsin B migrated as a single band of 39 kDa on SDS-PAGE. Most importantly, the molecule was not reactive with IgG in normal sera and, hence, could be employed in immunoblots or ELISA to specifically detect antibodies in schistosome-infected patients. Addition of oligosaccharides by P. pastoris is a potential drawback that needs to be considered before using P. pastoris-produced proteins as diagnostic reagents.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0022-1759
pubmed:author
pubmed:issnType
Print
pubmed:volume
304
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
151-7
pubmed:dateRevised
2007-8-13
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
De-glycosylation of Pichia pastoris-produced Schistosoma mansoni cathepsin B eliminates non-specific reactivity with IgG in normal human serum.
pubmed:affiliation
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Westbourne Street, Gore Hill, Sydney, NSW 2065, Australia.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't