Zhang X, Lok S H, Kon O L
Department of Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore.
Biochim Biophys Acta. 1998 Nov 27;1425(3):441-52. doi: 10.1016/s0304-4165(98)00095-6.
Hamster cell lines are common hosts for recombinant protein production, e.g. erythropoietin (Epo). Terminal sialylation of native human proteins is characteristically in both alpha-2,3 and alpha-2,6 linkage to galactose at the termini of N-linked oligosaccharides but only in alpha-2,3 linkage in recombinant proteins expressed in hamster cells which do not express alpha-2, 6-sialyltransferase (ST6GalI) (EC 2.4.99.1). This difference could alter the bioactivity of certain recombinant proteins. Chinese hamster ovary (CHO) cells stably transfected with human ST6GalI cDNA linked to the hamster metallothionein II promoter expressed highly inducible authentic ST6GalI activity. Untransfected CHO cells and CHO cells stably expressing ST6GalI cDNA when transfected with a human Epo cDNA expression cassette secreted immunoreactive Epo. Human Epo from singly transfected Pro-5 CHO cells induced significant reticulocytosis (7.00+/-1.58%; mean+/-S.D. % reticulocytes; control conditioned medium 3.04+/-1.29%; P<0.0024), whereas Epo from Pro-5 cells coexpressing ST6GalI elicited a more modest reticulocytosis (4.62+/-1.02%). Thus for recombinant human Epo, engineering CHO cells to express ST6GalI activity does not enhance Epo bioactivity in vivo in mice. The availability of CHO cells that express high levels of ST6GalI activity now enables systematic studies to determine the functional requirement for this form of sialylation in recombinant human proteins.
仓鼠细胞系是重组蛋白生产的常用宿主，例如促红细胞生成素（Epo）。天然人类蛋白的末端唾液酸化特征性地存在于N-连接寡糖末端与半乳糖的α-2,3和α-2,6连接中，但在不表达α-2,6-唾液酸转移酶（ST6GalI）（EC 2.4.99.1）的仓鼠细胞中表达的重组蛋白中仅存在于α-2,3连接中。这种差异可能会改变某些重组蛋白的生物活性。用与仓鼠金属硫蛋白II启动子相连的人ST6GalI cDNA稳定转染的中国仓鼠卵巢（CHO）细胞表达高度可诱导的真实ST6GalI活性。未转染的CHO细胞和稳定表达ST6GalI cDNA的CHO细胞在转染人Epo cDNA表达盒时分泌免疫反应性Epo。来自单转染的Pro-5 CHO细胞的人Epo诱导显著的网织红细胞增多（7.00±1.58%；网织红细胞平均±标准差%；对照条件培养基3.04±1.29%；P<0.0024），而来自共表达ST6GalI的Pro-5细胞的Epo引起更适度的网织红细胞增多（4.62±1.02%）。因此，对于重组人Epo，工程化CHO细胞以表达ST6GalI活性并不能增强其在小鼠体内的生物活性。现在，表达高水平ST6GalI活性的CHO细胞的可用性使得能够进行系统研究，以确定这种唾液酸化形式在重组人蛋白中的功能需求。
