In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans in Pichia pastoris
| Autor: | Roland Contreras, Wouter Vervecken, Nico Callewaert, Steven Geysens, Vladimir Kaigorodov, Kristof De Vusser |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Glycan
Glycosylation Recombinant Fusion Proteins Golgi Apparatus Mannose Endoplasmic Reticulum N-Acetylglucosaminyltransferases Applied Microbiology and Biotechnology Pichia Pichia pastoris symbols.namesake chemistry.chemical_compound Polysaccharides Mannosidases Humans chemistry.chemical_classification Ecology biology Endoplasmic reticulum Golgi apparatus Galactosyltransferases Physiology and Biotechnology biology.organism_classification Biochemistry chemistry symbols biology.protein Genetic Engineering Glycoprotein Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 70:2639-2646 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.70.5.2639-2646.2004 |
| Popis: | The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an α-1,2-mannosidase retained in the endoplasmic reticulum and N -acetylglucosaminyltransferase I and β-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides. |
| Databáze: | OpenAIRE |
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