Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells
Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells
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DOI：
10.1023/a:1026466408042
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发表时间：
1999-02-01
影响因子：
3
通讯作者：
Conradt, HS
中科院分区：
文献类型：
--
作者：
Grabenhorst, E;Schlenke, P;Conradt, HS
对许多天然糖蛋白及其来自哺乳动物宿主的重组对应物的分析已经揭示，糖基化多肽的基本寡糖结构和位点占据主要由蛋白质构象决定。（例如BHK-21和CHO-细胞）与糖基转移酶，核苷酸-糖基化酶和转运蛋白似乎足以保证重组蛋白的复合型糖基化，即使在高表达条件下也具有高度的末端α 2-3唾液酸化。一些人类组织特异性末端碳水化合物基序不能由这些细胞合成，因为它们缺乏适当的糖转移酶（例如α 1-3/4岩藻糖基转移酶、α 2-6唾液酸转移酶）。通过用编码末端人糖基转移酶的基因稳定转染对这些宿主进行糖基化工程改造，允许获得具有定制的糖基化的产物。（人组织特异性）糖基化。使用定点诱变，未糖基化的多肽可以通过转移糖基化结构域成功地转化为N-和/或O-糖蛋白糖蛋白和宿主细胞系的基因工程被认为提供了一种通用工具，以获得具有新的/新的氨基酸序列的治疗性糖产物。- 改善的体内性质，例如通过末端糖基化基序的合理设计引入特异性组织靶向信号。
The analysis of many natural glycoproteins and their recombinant counterparts from mammalian hosts has revealed that the basic oligosaccharide structures and the site occupancy of glycosylated polypeptides are primarily dictated by the protein conformation.The equipment of many frequently used host cells (e.g. BHK-21 and CHO-cells) with glycosyltransferases, nucleotide-sugar synthases and transporters appears to be sufficient to guarantee complex-type glycosylation of recombinant proteins with a high degree of terminal alpha 2-3 sialylation even under high expression conditions. Some human tissue-specific terminal carbohydrate motifs are not synthesized by these cells since they lack the proper sugar-transferring enzymes (e.g. alpha 1-3/4 fucosyltransferases, alpha 2-6 sialyltransferases). Glycosylation engineering of these hosts by stable transfection with genes encoding terminal human glycosyltransferases allows to obtain products with tailored (human tissue-specific) glycosylation in high yields.Using site-directed mutagenesis, unglycosylated polypeptides can be successfully converted in N- and/or O-glycoproteins by transferring glycosylation domains (consisting of 7-17 amino acids) from donor glycoproteins to different loop regions of acceptor proteins.The genetic engineering of glycoproteins and of host cell lines are considered to provide a versatile tool to obtain therapeutic glyco-products with novel/improved in-vivo properties, e.g. by introduction of specific tissue-targeting signals by a rational design of terminal glycosylation motifs.