Publikationsdatum:
2013-09-08
Beschreibung:
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.
Print ISSN:
0959-6658
Digitale ISSN:
1460-2423
Thema:
Biologie
,
Medizin
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