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 ␣-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and ␤-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.Most protein-based therapeutic agents produced in heterologous expression systems are glycosylated, a modification that is crucial for correct folding, stability, and bioactivity of the protein and influences its pharmacokinetic properties, such as tissue distribution and blood clearance. Glycoproteins with terminal sialic acids on their glycans persist longer in the blood than glycoproteins with terminal galactose, N-acetylglycosamine, or mannose residues because the latter compounds are cleared rapidly via receptors in the liver and on reticuloendothelial cells (e.g., the asialoglycoprotein receptor and the mannose receptor) (10,20,30,31). In addition, glycan structures produced in nonhuman cells can cause immune reactions, as exemplified by the reaction against xenografts of porcine origin; these reactions are primarily caused by the presence of ␣-galactose on the glycoproteins (7). Another example is the immune reaction against glycoproteins from yeast, which results from the presence of ␣-1,3-mannose, ␤-linked mannose, and/or phosphate residues in either a phosphomonoester or phosphodiester linkage (1, 32). Consequently, recombinant glycoproteins produced for therapeutic applications should be expressed in heterologous hosts that produce protein-linked oligosaccharides that closely resemble those of humans.

The gene for the open reading frame YER005w that is homologous to yeast Golgi GDPase encoded by the GDA1 gene was cloned and named YND1. It encodes a 630-amino acid protein that contains a single transmembrane region near the carboxyl terminus. The overexpression of the YND1 gene in the gda1 null mutant caused a significant increase in microsomal membranebound nucleoside phosphatase activity with a luminal orientation. The activity was equally high toward ADP/ ATP, GDP/GTP, and UDP/UTP and ϳ50% less toward CDP/CTP and thiamine pyrophosphate, but there was no activity toward GMP, indicating that the Ynd1 protein belongs to the apyrase family. This substrate specificity is different from that of yeast GDPase, but similar to that of human Golgi UDPase. The ⌬ynd1 mutant cells were defective in O-and N-linked glycosylation in the Golgi compartments. The overexpression of the YND1 gene complemented some glycosylation defects in ⌬gda1 disruptants, suggesting a partially redundant function of yeast apyrase and GDPase. From these results and the phenotype of the ⌬ynd1⌬gda1 double deletion showing a synthetic effect, we conclude that yeast apyrase is required for Golgi glycosylation and cell wall integrity, providing the first direct evidence for the in vivo function of intracellular apyrase in eukaryotic cells.

Background: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs.

Glycosylation is an important issue in heterologous protein production for therapeutic applications. Glycoproteins produced in Pichia pastoris contain high mannose glycan structures that can hamper downstream processing, might be immunogenic, and cause rapid clearance from the circulation. This chapter describes a method that helps solving these glycosylation-related problems by inactivation of OCH1, overexpression of an HDEL-tagged mannosidase, and overexpression of a Kre2/GlcNAc-transferase I chimeric enzyme. Different plasmids are described as well as glycan analysis methods.