(570d) Engineering the N-Glycosylation Pathway in Pichia Pastoris for the Expression of Glycoprotein Hormones

Yeast cells serve as model systems for the study and understanding of eukaryotic cell functioning. They are also the ideal choice for the expression of proteins of therapeutic importance. Around 70% of the therapeutic proteins under preclinical and clinical development are glycosylated and hence an increasing demand for expression system with the ability to perform post translational modification. N-glycosylation is a post translational modification that occurs in the endoplasmic reticulum (ER) and the Golgi complex. The end product of the N-glycosyaltion is observed to be different among species. The N-glycan structure is important for proper folding and activity of the protein, its serum half-life and also defines its immunogenicity. Hence having an expression system with human like N-glycosylation is imperative.

The yeast Pichia pastoris is gaining increasing attention as a protein production host due to the ease of transformation, ability to perform post translational modification, growth on defined media to high cell densities and scalable fermentations, low concentration of host proteins secreted into the media enabling easy downstream processing and high expression rate. Many of the therapeutic proteins expressed in Pichia are currently in clinical trials. Yeast cells are known to have the high mannose type oligosaccharide chain attached to the proteins (Man_9-20GlcNAc₂). In contrast, the human N-glycosylation is of the mixed or hybrid type (NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂).

Engineerring the N-glycosylation pathway in the yeast would involve introduction of the repository of the enzymes from the higher eukaryotic pathway and eliminating the non-human glycosylation reactions. The first step towards modification of the pathway involves the disruption of the endogeneous glycosyltransferase gene (OCH1) that initalizes the addition of excess mannose to oligosaccharide chain. Furthur heterologous glycosidases and glycosyltransferases have to be introduced. Given that these ezymatic reactions occur in a seqential manner with the product of one enzyme acting as the substrate of the next, proper targeting of these enzymes to the cisternae of Golgi complex is important. A host of enzymes from higher eukaryotic cells and a proper targeting sequence fused to the protein were used to create a host cell expressing proteins with human like N-glycans.

The host thus generated was used to express the glycoprotien hormones, Follicle Stimulating Hormone (FSH) and human Chorionic Gonadotropin (hCG). FSH and hCG controls the reproductive functions, gametogenesis and are important for the maintenance of pregnancy in humans. Both the hormones are heterodimeric molecules comprising of an α-subunit and a β-subunit in non-covalent association with two N-glycosylation sites on each subunit. Purified hormones are important for the study of stucture - function relationships, role of N-glycoslation in protein folding, treatment of in-fertile patients suffering from gonadotropin deficiency and in-vitro fertilization (IVF). In the present study we report the expression and purification of FSH and hCG under the costitutively expressing GAP promoter in the glycoengineered Pichia pastoris. The cells were grown to high densities of 540g/L in fermentor culture and the protein expression was found to be 51mg/L (FSH) and 37mg/L (hCG). The expressed proteins were purified by a two step chromatography and characterized by various biochemical assays. The purified hormones were found to have the modified glycan structure and showed activity in Radioreceptor assay with the cognate receptor. The recombinant hormones were administered to animals for testing their in vivo bioactivity and found to be biologically active. FSH activity was determined by the increase in ovarian and uterine weight in female rats and hCG activity was determined by the testosterone response in male rats and bonnet monkeys.  

Abbreviations: NANA - Sialic Acid, Gal - Galactose, GlcNAc - N-acetylglucoseamine, Man - Mannose, GAP - glyceraldehyde-3-phosphate dehydrogenase