Use of high-performance anion exchange chromatography with pulsed amperometric detection for O-glycan determination in yeast.
Nat Protoc
; 3(6): 1026-31, 2008.
Article
em En
| MEDLINE
| ID: mdl-18546597
ABSTRACT
O-glycosylation is a post-translational protein modification that occurs in all eukaryotes. Yeasts have received increasing attention as a host for therapeutic protein production because of their ability to secrete high levels of recombinant protein. Because yeasts such as Pichia pastoris have been shown to O-glycosylate some proteins with varying effects on protein function, it is important to elucidate the nature of this modification. Methods that characterize O-glycosylation on a qualitative and quantitative basis are thus important when considering yeast as a host for therapeutic protein production. This protocol describes the release of O-glycans from a protein sample by -elimination under alkaline conditions using sodium borohydride and sodium hydroxide. The released O-linked oligosaccharides are subsequently processed and then separated by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). An estimation of O-glycan molar occupancy and average O-mannose chain length is ultimately derived. This protocol requires approximately 3 d for completion. This method provides an assessment of O-glycosylation and allows one to correlate the effect of O-glycosylation on protein properties.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Leveduras
/
Cromatografia por Troca Iônica
/
Micologia
Tipo de estudo:
Diagnostic_studies
/
Guideline
/
Qualitative_research
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Estados Unidos