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

Abstract

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, PGAP and PAOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI‐MS. In the samples expressed with PGAP 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 PAOX1. The results indicate that the O‐glycosylation level is markedly higher when the protein is produced in a methanol‐based expression system.

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation

“…These latter are due to the presence of a serine/threonine rich C-terminal region (76% serine/threonines content) with, interestingly, Cht3 as a whole being composed of as much as 103 serines (constituting 18.7% of the total amino acid content) and 98 threonines (17.8% of the total). Importantly, P. pastoris is also capable of carrying out both N-and O-linked glycosylation of secreted proteins [43,44] and in this study recombinant Cht3 was confirmed as being mainly O-glycosylated, similar to that reported for other C. albicans…”

Improved Production of Recombinant Myrosinase in Pichia pastoris

“…Proteins are correctly folded and glycosylated in a pattern similar to mammalian cells. P. pastoris is able to perform O- and N-glycosylation [ 5 ], the latter having been analyzed for many years and, in the end, its humanization was achieved through glycoengineering [ 6 ]. Moreover, P. pastoris is a suitable system for the production of secreted proteins, because it not only processes signal peptides correctly but also secretes a limited amount of endogenous proteins to the culture supernatant [ 1 ].…”

Current advances of Pichia pastoris as cell factories for production of recombinant proteins

“…However, the removal of N-glycosylation didn’t make great changes on the secondary structure of proteins but tertiary structure showed differences ( Wang et al, 2018 ). Another recent study found that the degree of O-glycosylation was remarkably higher when the protein was induced by methanol compared with glucose ( Radoman et al, 2021 ), but details of machinery required to be investigated later.…”