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The Degree and Length of O ‐Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type
Author(s) -
Radoman Bojana,
GrünwaldGruber Clemens,
Schmelzer Bernhard,
Zavec Domen,
Gasser Brigitte,
Altmann Friedrich,
Mattanovich Diethard
Publication year - 2021
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.202000266
Subject(s) - pichia pastoris , glycosylation , yeast , biochemistry , recombinant dna , pichia , glycoprotein , heterologous , glycan , n linked glycosylation , biology , chemistry , gene
The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N‐linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O ‐glycosylation. O ‐glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O ‐mannosylation profiles in P. pastoris . Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O ‐glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P GAP and P AOX1 . After purification and protein digestion, glycopeptides are analyzed by LC/ESI‐MS. In the samples expressed with P GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P AOX1 . The results indicate that the O ‐glycosylation level is markedly higher when the protein is produced in a methanol‐based expression system.