In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris
In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris
复制标题
DOI：
10.1128/aem.70.5.2639-2646.2004
复制
发表时间：
2004-05-01
影响因子：
4.4
通讯作者：
Contreras, R
中科院分区：
文献类型：
--
作者：
Vervecken, W;Kaigorodov, V;Contreras, R
毕赤酵母n -糖基化途径与人类细胞中的途径仅部分同源。在高尔基体中，人类细胞合成复杂的低聚糖，而毕赤酵母细胞形成的甘露糖结构可含有多达40个甘露糖残基。分泌的糖蛋白的高糖基化阻碍了异源表达的糖蛋白的下游加工，并导致基于蛋白质的治疗药物的产生，这些药物由于末端甘露糖残基的存在而迅速从血液中清除。在这里，我们描述了工程的P. pastoris n -糖基化途径，以产生非高糖基化杂交聚糖。这是通过OCH1的失活和保留在内质网中的α -1,2-甘露糖苷酶以及保留在高尔基体中的n -乙酰氨基葡萄糖转移酶I和β -1,4-半乳糖转移酶的过表达来实现的。该工程菌株在其糖蛋白上合成了一个非唾液化的杂交型n -连接低聚糖结构。我们开发的程序允许任何酵母表达菌株的聚糖工程，并且可以产生高达90%的均匀蛋白连接低聚糖。
The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an alpha-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and beta-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.