High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains -: selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active N-15-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose-glycerol-methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man(8-15)GlcNAc(2)). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Man(up-to-30)GlcNAc(2)). The latter finding made the X-33 strain not very suitable for generating N-15-labeled material. Therefore, N-15-phCG was expressed in the GS115 strain using the new optimized protocol. The (15)sN-enrichment was evaluated by N-15-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that N-15-phCG/GS115 had the same folding as urinary hCG. Furthermore, N-15-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.