Graz University of Technology, Institute of Molecular Biotechnology, Graz, Austria.
Sci Rep. 2013 Nov 20;3:3279. doi: 10.1038/srep03279.
The yeast Pichia pastoris is a common host for the recombinant production of biopharmaceuticals, capable of performing posttranslational modifications like glycosylation of secreted proteins. However, the activity of the OCH1 encoded α-1,6-mannosyltransferase triggers hypermannosylation of secreted proteins at great heterogeneity, considerably hampering downstream processing and reproducibility. Horseradish peroxidases are versatile enzymes with applications in diagnostics, bioremediation and cancer treatment. Despite the importance of these enzymes, they are still isolated from plant at low yields with different biochemical properties. Here we show the production of homogeneous glycoprotein species of recombinant horseradish peroxidase by using a P. pastoris platform strain in which OCH1 was deleted. This och1 knockout strain showed a growth impaired phenotype and considerable rearrangements of cell wall components, but nevertheless secreted more homogeneously glycosylated protein carrying mainly Man8 instead of Man10 N-glycans as a dominant core glycan structure at a volumetric productivity of 70% of the wildtype strain.
毕赤酵母是生物制药重组生产的常用宿主，能够对分泌蛋白进行翻译后修饰，如糖基化。然而，OCH1 编码的α-1,6-甘露糖基转移酶的活性会导致分泌蛋白发生高度不均一的甘露糖化，严重影响下游处理和重现性。辣根过氧化物酶是一种多功能酶，在诊断、生物修复和癌症治疗中有应用。尽管这些酶很重要，但它们仍然是从植物中以低产量分离得到的，具有不同的生化特性。在这里，我们展示了通过使用缺失 Och1 的毕赤酵母平台菌株来生产重组辣根过氧化物酶的均一糖蛋白。这种 och1 敲除菌株表现出生长受损的表型和细胞壁成分的显著重排，但仍能分泌更均匀糖基化的蛋白，主要携带 Man8 而不是 Man10 N-聚糖作为主要核心糖结构，其比活产量为野生型菌株的 70%。
