ArticleGlycoconjugate journal2008
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.
Article in Glycoconjugate journal, 2008. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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11 citing papers in PubMed, 33 citations in OpenAlex.
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- Influence of codon optimization, promoter, and strain selection on the heterologous production of a β-fructofuranosidase from Aspergillus fijiensis ATCC 20611 in Pichia pastoris.Folia microbiologica · 2022Article
- Improved Production of Recombinant Myrosinase inInternational journal of molecular sciences · 2021Article
- Comparative genome-scale analysis of Pichia pastoris variants informs selection of an optimal base strain.Biotechnology and bioengineering · 2020Article
- Genomic diversity and meiotic recombination among isolates of the biotech yeast Komagataella phaffii (Pichia pastoris).Microbial cell factories · 2019Article
- Expression of a RecombinantFrontiers in microbiology · 2018Article
- Detection of distinct glycosylation patterns on human γ-glutamyl transpeptidase 1 using antibody-lectin sandwich array (ALSA) technology.BMC biotechnology · 2014Article
- Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain.Journal of biomolecular NMR · 2011Article
- Invasion of Trypanosoma cruzi into host cells is impaired by N-propionylmannosamine and other N-acylmannosamines.Glycoconjugate journal · 2011Article
- High yield expression of an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture.Microbial cell factories · 2010Article
- Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins.Journal of biomolecular NMR · 2010Review
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Authors and funding
9 authors at 3 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-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 (Man8-15GlcNAc2). 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 (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.
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