De-glycosylation of Pichia pastoris-produced Schistosoma mansoni cathepsin B eliminates non-specific reactivity with IgG in normal human serum

Stack, CM; Dalton, JP; Cunneen, M; Donnelly, S

De-glycosylation of Pichia pastoris-produced Schistosoma mansoni cathepsin B eliminates non-specific reactivity with IgG in normal human serum

Stack, CM Dalton, JP Cunneen, M Donnelly, S

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Publication Type:: Journal Article
Citation:: Journal of Immunological Methods, 2005, 304 (1-2), pp. 151 - 157
Issue Date:: 2005-09-01

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Field	Value	Language
dc.contributor.author	Stack, CM	en_US
dc.contributor.author	Dalton, JP	en_US
dc.contributor.author	Cunneen, M	en_US
dc.contributor.author	Donnelly, S https://orcid.org/0000-0003-2005-3698	en_US
dc.date.available	2005-07-12	en_US
dc.date.issued	2005-09-01	en_US
dc.identifier.citation	Journal of Immunological Methods, 2005, 304 (1-2), pp. 151 - 157	en_US
dc.identifier.issn	0022-1759	en_US
dc.identifier.uri	http://hdl.handle.net/10453/4494
dc.description.abstract	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. © 2005 Elsevier B.V. All rights reserved.	en_US
dc.relation.ispartof	Journal of Immunological Methods	en_US
dc.relation.isbasedon	10.1016/j.jim.2005.07.019	en_US
dc.subject.classification	Immunology	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	Schistosoma mansoni	en_US
dc.subject.mesh	Pichia	en_US
dc.subject.mesh	Schistosomiasis mansoni	en_US
dc.subject.mesh	Cathepsin B	en_US
dc.subject.mesh	Immunoglobulin G	en_US
dc.subject.mesh	Immunoblotting	en_US
dc.subject.mesh	Enzyme-Linked Immunosorbent Assay	en_US
dc.subject.mesh	Mutagenesis, Site-Directed	en_US
dc.subject.mesh	Cross Reactions	en_US
dc.subject.mesh	Glycosylation	en_US
dc.title	De-glycosylation of Pichia pastoris-produced Schistosoma mansoni cathepsin B eliminates non-specific reactivity with IgG in normal human serum	en_US
dc.type	Journal Article
utslib.citation.volume	1-2	en_US
utslib.citation.volume	304	en_US
utslib.for	1107 Immunology	en_US
utslib.for	1108 Medical Microbiology	en_US
utslib.for	1113 Opthalmology and Optometry	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Life Sciences
utslib.copyright.status	closed_access
pubs.issue	1-2	en_US
pubs.publication-status	Published	en_US
pubs.volume	304	en_US

Abstract:

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. © 2005 Elsevier B.V. All rights reserved.

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http://hdl.handle.net/10453/4494