Seo N S, Hollister J R, Jarvis D L
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, 82071-3944, USA.
Protein Expr Purif. 2001 Jul;22(2):234-41. doi: 10.1006/prep.2001.1432.
The baculovirus-insect cell expression system is widely used to produce recombinant mammalian glycoproteins, but the glycosylated end products are rarely authentic. This is because insect cells are typically unable to produce glycoprotein glycans containing terminal sialic acid residues. In this study, we examined the influence of two mammalian glycosyltransferases on N-glycoprotein sialylation by the baculovirus-insect cell system. This was accomplished by using a novel baculovirus vector designed to express a mammalian alpha2,6-sialyltransferase early in infection and a new insect cell line stably transformed to constitutively express a mammalian beta1,4-galactosyltransferase. Various biochemical assays showed that a foreign glycoprotein was sialylated by this virus-host combination, but not by a control virus-host combination, which lacked the mammalian glycosyltransferase genes. Thus, this study demonstrates that the baculovirus-insect cell expression system can be metabolically engineered for N-glycoprotein sialylation by the addition of two mammalian glycosyltransferase genes.
杆状病毒-昆虫细胞表达系统被广泛用于生产重组哺乳动物糖蛋白，但糖基化终产物很少是真实的。这是因为昆虫细胞通常无法产生含有末端唾液酸残基的糖蛋白聚糖。在本研究中，我们通过杆状病毒-昆虫细胞系统研究了两种哺乳动物糖基转移酶对N-糖蛋白唾液酸化的影响。这是通过使用一种新型杆状病毒载体来实现的，该载体设计用于在感染早期表达哺乳动物α2,6-唾液酸转移酶，以及一种新的稳定转化的昆虫细胞系，该细胞系可组成性表达哺乳动物β1,4-半乳糖基转移酶。各种生化分析表明，一种外源糖蛋白可被这种病毒-宿主组合唾液酸化，但缺乏哺乳动物糖基转移酶基因的对照病毒-宿主组合则不能。因此，本研究表明，通过添加两个哺乳动物糖基转移酶基因，杆状病毒-昆虫细胞表达系统可进行代谢工程改造以实现N-糖蛋白的唾液酸化。
