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Production of sialylated O-linked glycans in Pichia pastoris
- Source :
- Glycobiology. 23:1192-1203
- Publication Year :
- 2013
- Publisher :
- Oxford University Press (OUP), 2013.
-
Abstract
- The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N-linked glycans but up to now no one has addressed engineering the O-linked glycosylation pathway. Typically, O-linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O-linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O-linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein-O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, resulted in the capping of the single O-linked mannose residues with N-acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O-linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N-linked glycosylated biotherapeutics to include molecules possessing O-linked glycans.
- Subjects :
- Mannosidase
chemistry.chemical_classification
Glycan
Glycosylation
biology
Mannose
Protein Engineering
biology.organism_classification
Biochemistry
Pichia
Yeast
Pichia pastoris
carbohydrates (lipids)
chemistry.chemical_compound
Residue (chemistry)
Enzyme
Metabolic Engineering
chemistry
alpha-Mannosidase
biology.protein
Subjects
Details
- ISSN :
- 14602423 and 09596658
- Volume :
- 23
- Database :
- OpenAIRE
- Journal :
- Glycobiology
- Accession number :
- edsair.doi.dedup.....ef85cbf2997467f77dbc6ad086fca73c