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.

中文翻译：

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毕赤酵母中产生的重组蛋白的O-糖基化程度和长度取决于该蛋白的性质和过程类型

甲基营养酵母巴斯德毕赤酵母被认为是生产异源蛋白的有效宿主。尽管N.连接的蛋白糖基化在巴斯德毕赤酵母中具有很好的特征，但是对O-糖基化模式的了解却很少。巴斯德毕赤酵母产生的O-聚糖由短的线性甘露糖链组成，在重组生物制药的情况下，它可以触发人类的免疫反应。本研究旨在揭示不同培养策略对巴斯德毕赤酵母O-甘露糖基化谱的影响。分析了由不同巴斯德毕赤酵母菌株产生的16种不同模型蛋白的蛋白质O-糖基化谱。根据获得的数据，选择人类血清白蛋白（HSA）以通过使用常见启动子P _GAP和P _AOX1在快速和缓慢生长的补料分批发酵中生产。纯化和蛋白质消化后，通过LC / ESI-MS分析糖肽。在以P表示样品_GAP发现的糖基化的程度是当使用慢生长速率稍高，无论生产菌株的效率。最高的糖基化强度观察到HSA产生的P _AOX1。结果表明，在基于甲醇的表达系统中生产蛋白质时，O-糖基化水平明显更高。