Glyco-engineering of moss lacking plant-specific sugar residues
Glyco-engineering of moss lacking plant-specific sugar residues
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DOI：
10.1055/s-2005-837653
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发表时间：
2005-05-01
期刊：
影响因子：
3.9
通讯作者：
Decker, EL
中科院分区：
文献类型：
--
作者：
Huether, CM;Lienhart, O;Decker, EL
在植物中商业化生产来自人类的复杂药物蛋白目前受到植物和人类之间蛋白质N-糖基化模式差异的限制。一方面，植物特异性α（1，3）-岩藻糖和β（1，2）-木糖残基显示出具有强的免疫原性潜力。另一方面，末端β（1，4）-半乳糖是药学相关蛋白质的N-聚糖上常见的糖，例如，在植物N-聚糖结构中缺少抗体。为了安全和灵活地生产药物蛋白，植物蛋白N-糖基化的人源化是必不可少的。在这里，我们提出了一种结合避免植物特异性和引入人类聚糖结构的方法。创建苔藓小立碗藓的转基因菌株，其中通过在两种植物基因中靶向插入人β（1，4）-半乳糖基转移酶编码序列（敲入）来敲除α（1，3）-岩藻糖基转移酶和β（1，2）-木糖基转移酶基因。转基因缺乏α（1，3）-岩藻糖和β（1，2）-木糖残基，而β（1，4）-半乳糖残基出现在蛋白质N-聚糖上。尽管有这些显著的生物化学变化，但在标准培养条件下，植物的总体形态与野生型没有差异。此外，糖工程植物分泌的瞬时表达的重组人蛋白，血管内皮生长因子，在相同的浓度为未修饰的苔藓，表明糖基化的变化并没有损害苔藓的分泌途径。本文提出的组合敲除/敲入方法导致新一代工程化苔藓，并朝向具有人源化N-糖基化谱的正确加工的药物蛋白质的安全和灵活的生产。
The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific alpha(1,3)-fucose and beta(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal beta(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the alpha(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human beta(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked alpha(1,3)-fucose and beta(1,2)-xylose residues, whereas beta(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretary pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.