Kim Yeon Kyu, Shin Hwa Sung, Tomiya Noboru, Lee Yuan C, Betenbaugh Michael J, Cha Hyung Joon
Department of Chemical Engineering and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Korea.
Biotechnol Bioeng. 2005 Nov 20;92(4):452-61. doi: 10.1002/bit.20605.
Schneider 2 (S2) cells from Drosophila melanogaster have been used as a plasmid-based, non-lytic expression system for foreign proteins. Here, a plasmid encoding the human erythropoietin (hEPO) gene fused with a hexahistidine (His(6)) tag under the control of the Drosophila metallothionein (MT) promoter was stably transfected into Drosophila S2 cells. After copper sulfate induction, transfected S2 cells were found to secrete hEPO with a maximum expression level of 18 mg/L and a secretion efficiency near 98%. The secreted hEPO from Drosophila S2 had an apparent molecular weight of about 23-27 kDa which was significantly lower than a recombinant hEPO expressed in Chinese hamster ovary (CHO) cells (about 36 kDa). N-glycosidase F digestion almost completely eliminated the difference and resulted in the same molecular weight ( approximately 20 kDa) of de-N-glycosylated hEPO proteins. These data suggest that recombinant hEPO from S2 cells was modified with smaller N-glycans. Subsequently, the major N-glycans were identified following glycoamidase A digestion, labeling with 2-aminopyridine (PA), and two-dimensional high-performance liquid chromatography (HPLC) analysis in concert with exoglycosidase digestion. This analysis of N-glycans revealed that hEPO was modified to include paucimannosidic glycans containing two or three mannose residues with or without core fucose. A similar glycosylation pattern was observed on a recombinant human transferrin expressed in S2 cells. These results provide a detailed analysis of multiple N-glycan structures produced in a Drosophila cell line that will be useful in the subsequent application of these cells for the generation of heterologous glycoproteins.
果蝇的 Schneider 2（S2）细胞已被用作基于质粒的外源蛋白非裂解表达系统。在此，将一个编码人促红细胞生成素（hEPO）基因且与六聚组氨酸（His(6)）标签融合的质粒，在果蝇金属硫蛋白（MT）启动子的控制下，稳定转染到果蝇 S2 细胞中。经硫酸铜诱导后，发现转染的 S2 细胞分泌 hEPO，最大表达水平为 18 mg/L，分泌效率接近 98%。果蝇 S2 细胞分泌的 hEPO 的表观分子量约为 23 - 27 kDa，显著低于在中国仓鼠卵巢（CHO）细胞中表达的重组 hEPO（约 36 kDa）。N - 糖苷酶 F 消化几乎完全消除了这种差异，使去 N - 糖基化的 hEPO 蛋白分子量相同（约 20 kDa）。这些数据表明，S2 细胞中的重组 hEPO 被较小的 N - 聚糖修饰。随后，在进行糖酰胺酶 A 消化、2 - 氨基吡啶（PA）标记、二维高效液相色谱（HPLC）分析并结合外切糖苷酶消化后，鉴定出了主要的 N - 聚糖。这种 N - 聚糖分析表明，hEPO 被修饰为包含含有两个或三个甘露糖残基且有或没有核心岩藻糖的寡甘露糖型聚糖。在 S2 细胞中表达的重组人转铁蛋白上也观察到了类似的糖基化模式。这些结果对果蝇细胞系中产生的多种 N - 聚糖结构进行了详细分析，这将有助于这些细胞随后用于生成异源糖蛋白。
