Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
Enzyme Microb Technol. 2013 Aug 15;53(3):170-3. doi: 10.1016/j.enzmictec.2013.03.020. Epub 2013 Apr 1.
Human 90K (h90K; Mac-2-binding protein) glycoprotein is a potential pharmaceutical due to its inhibitory activity against cancer metastasis and expansion. Here, h90K glycoprotein was produced in insect Drosophila S2 cell system, and its N-glycan pattern was analyzed. A plasmid encoding h90K gene, fused with a hexahistidine tag under the control of Drosophila metallotionein promoter, was stably transfected into S2 cells. After copper sulfate induction, transfected S2 cells secreted recombinant h90K at a good expression level of 28mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of ∼78kDa which was much smaller than that (∼97kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at ∼60kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (∼18kDa) of N-glycans of the natural h90K. Through detail analyses using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the S2-derived recombinant h90K was confirmed that it has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major (∼79%) N-glycans.
人 90K（h90K；Mac-2 结合蛋白）糖蛋白是一种有潜力的药物，因为它具有抑制癌症转移和扩张的活性。在这里，h90K 糖蛋白在昆虫果蝇 S2 细胞系统中产生，并分析了其 N-聚糖模式。一个编码 h90K 基因的质粒，在果蝇金属硫蛋白启动子的控制下融合了六组氨酸标签，被稳定转染到 S2 细胞中。在硫酸铜诱导后，转染的 S2 细胞在 150 毫升摇瓶培养中以 28mg/L 的良好表达水平分泌重组 h90K。纯化的重组 h90K 表现出约 78kDa 的明显分子量，明显小于天然 h90K 的分子量（约 97kDa）。由于去 N-糖基化的 h90K 出现在约 60kDa 的蛋白带，因此表明 S2 细胞中的重组 h90K 具有较小的 N-聚糖，其分子量约为天然 h90K 的 N-聚糖的一半（约 18kDa）。通过使用高效液相色谱和基质辅助激光解吸/离子化飞行时间质谱的详细分析，证实了 S2 衍生的重组 h90K 具有简单的低聚糖结构，含有两个或三个甘露糖残基，核心岩藻糖作为主要（约 79%）的 N-聚糖。
