BACKGROUND Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N-Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N-glycoproteins with defined N-glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N-glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N-glycan structures and the N-glycosylation microheterogeneities between batches. RESULTS In this study, we constructed a Pichia pastoris (Komagataella phaffii) expression system producing truncated N-GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N-glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N-glycans and the N-GlcNAc modification on the glycosite Asn297 was confirmed via Mass Spectrometry. CONCLUSION This strategy develops a simple glycoengineered yeast expression system to produce N-GlcNAc modified proteins, which could be further extended to different N-glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.

中文翻译：

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巴斯德毕赤酵母中重组N-GlcNAc蛋白的均质生产和表征。

背景技术治疗性糖蛋白在生物药物市场中已占据极其重要的位置。蛋白药物的N-糖基化有助于它们保持最佳构象并影响其结构稳定性，血清半衰期和生物学效率。因此，具有确定的N-聚糖的均质N-糖蛋白在其临床治疗中的应用是必不可少的。但是，获得均质N-聚糖仍然存在一些障碍，例如由于普遍使用哺乳动物细胞表达系统，非人源化N-聚糖结构以及批次之间的N-糖基化微异质性而导致的高生产成本。结果在这项研究中，我们通过引入对高甘露糖型N-糖具有强大水解活性的ENGase同工型（Endo-T），构建​​了一种被截短的N-GlcNAc修饰的重组蛋白的巴斯德毕赤酵母（Komagataella phaffii）表达系统。结果表明，Endo-T在不同亚细胞部分（如内质网（ER），高尔基体或细胞膜）中的位置会影响其水解效率。当Endo-T在高尔基体中表达时，分泌的IgG1-Fc区几乎可以被完全截断的N-聚糖有效地产生，并且通过质谱确认了糖位点Asn297上的N-GlcNAc修饰。结论该策略开发了一种简单的糖工程化酵母表达系统，可产生N-GlcNAc修饰的蛋白质，可以进一步扩展到不同的N-聚糖结构。该系统将为大规模生产增加的新型糖蛋白药物提供一个前瞻性平台。