稳定的人O-聚糖核心2β-1,6-N-乙酰氨基葡萄糖转移酶在Sf9昆虫细胞中的表达。
Expression of stable human O-glycan core 2 beta-1,6-N-acetylglucosaminyltransferase in Sf9 insect cells.
作者信息
Toki D, Sarkar M, Yip B, Reck F, Joziasse D, Fukuda M, Schachter H, Brockhausen I
机构信息
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
出版信息
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):63-9. doi: 10.1042/bj3250063.
Abstract
UDP-GlcNAc:Galbeta1-3GalNAc-R (GlcNAc to GalNAc) beta-1, 6-N-acetylglucosaminyltransferase (C2GnT) catalyses the formation of O-glycan core 2. Purification and characterization of C2GnT from natural sources has been hampered by the instability of this enzyme. We have been able to prepare a stable partly purified recombinant human C2GnT by expression of a truncated form of the enzyme in the baculovirus/Spodoptera frugiperda 9 (Sf9) insect cell system. C2GnT activity was secreted into the Sf9 culture medium (15 pmol/min per microl; approx. 0.2 mg/l) and was stable at 4 degrees C either in solution or after lyophilization. Endoglycosidase H and N-glycanase F treatment of the radiolabelled C2GnT indicated the presence of N-glycans at both potential N-glycosylation sites. The elimination of one or both of the two potential N-glycosylation sites or treatment of the virus-infected insect cells with tunicamycin resulted in loss of enzyme activity due in part to protein degradation.
摘要
UDP-N-乙酰葡糖胺：β-1,3-半乳糖基-N-乙酰半乳糖胺-R（GlcNAc到GalNAc）β-1,6-N-乙酰葡糖胺基转移酶（C2GnT）催化O-聚糖核心2的形成。由于该酶的不稳定性，从天然来源纯化和表征C2GnT受到了阻碍。我们通过在杆状病毒/草地贪夜蛾9（Sf9）昆虫细胞系统中表达该酶的截短形式，成功制备了一种稳定的部分纯化的重组人C2GnT。C2GnT活性分泌到Sf9培养基中（每微升15皮摩尔/分钟；约0.2毫克/升），并且在4℃下无论是在溶液中还是冻干后都很稳定。对放射性标记的C2GnT进行内切糖苷酶H和N-聚糖酶F处理表明，在两个潜在的N-糖基化位点均存在N-聚糖。消除两个潜在N-糖基化位点中的一个或两个，或用衣霉素处理病毒感染的昆虫细胞，都会导致酶活性丧失，部分原因是蛋白质降解。
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