哺乳动物细胞中糖链结构的改造  

Remolding N-Glycan Structure in HEK293 Cells

作　　者：金则成 喜多岛敏彦 藤田盛久 高晓冬[1] JIN Zecheng;TOSHIHIKO Kitajima;MORIHISA Fujita;GAO Xiaodong(School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与生物技术学报》2020年第2期39-46,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31400693);中央高校基本科研业务费项目(JUSRP51508)。

摘　　要：由于蛋白质需要正确的折叠和糖基化修饰,目前生物药用蛋白质主要通过哺乳动物细胞生产。然而利用哺乳动物细胞生产医药蛋白质也面临着高成本、糖链不均一等问题。本研究中,我们构建了敲除高尔基体α-1,2-甘露糖苷酶Ⅰ的HEK293细胞株,其中MAN1A1和MAN1A2双敲除细胞株D-KO35中蛋白质上高甘露糖型N-比例增加。在这株双敲细胞中,溶酶体膜蛋白LAMP2上的糖链对糖链内切酶-H处理敏感,证明蛋白上N-糖链结构以高甘露糖型结构为主。研究结果表明,在哺乳动物细胞中敲除高尔基体α-1,2-甘露糖苷酶的细胞株,可用于生产均一化N-糖链结构的重组糖蛋白。Many of biopharmaceutical proteins are produced by mammalian cells because the proteins require correct folding and glycosylation.However,production of recombinant proteins in mammalian cells has some issues to be solved,including higher manufacturing cost and heterogeneity of glycans attached to the proteins.To overcome the issue,we here established a HEK293 cell lines disrupted Golgi alpha-1,2-mannosidase I.In double-KO cell line,which was knocked out both MAN1A1 and MAN1A2,the high mannose-type N-linked glycans were increased,whereas complex type glycans was greatly reduced.The glycans of an endogenous protein LAMP2 were sensitive to endoglycosidase-H treatment in double-KO cells,indicating that most of glycans on the proteins were high mannose-type glycans.Our results suggest that disruption of alpha-1,2-mannosidases in mammalian cells,especially double-KO cells,is useful for production of recombinant proteins with high mannose-type glycans.

关 键 词：N-糖基化 α-1 2-甘露糖苷酶 糖蛋白 HEK293细胞 

分 类 号：Q786[生物学—分子生物学]