Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells
Production and N-glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells
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DOI：
10.1002/bit.20605
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发表时间：
2005-11-20
影响因子：
3.8
通讯作者：
Cha, HJ
中科院分区：
文献类型：
--
作者：
Kim, YK;Shin, HS;Cha, HJ
黑腹果蝇的Schneider 2(S2)细胞已被用作外源蛋白的非裂解表达系统。将人促红细胞生成素(HEPO)基因与果蝇金属硫蛋白(MT)启动子控制下的六氢组氨酸(His(6))标签融合，稳定地导入果蝇S2细胞。经硫酸铜诱导后，转染组S2细胞分泌hEPO，最高表达水平为18 mg/L，分泌效率接近98%。果蝇S2分泌的hEPO表观分子量约为23 kDa，与27 kDa相近，明显低于中国仓鼠卵巢(CHO)细胞表达的重组hEPO(约36 kDa)。N-糖苷酶F消化几乎完全消除了这种差异，得到了相同的分子量(约20 kDa)的去N-糖基化的hEPO蛋白。这些数据表明，来自S2细胞的重组hEPO被较小的N-糖链修饰。随后，通过糖胺酶A消化、2-氨基吡啶(PA)标记和结合外切糖苷酶消化的二维高效液相(HPLC)分析，鉴定了主要的N-糖链。对N-糖链的分析表明，hEPO被修饰为包括含有两个或三个甘露糖残基的稀糖甘露糖苷，带有或不带有核心岩藻糖。在S2细胞中表达的重组人转铁蛋白也有类似的糖基化模式。这些结果提供了对果蝇细胞系中产生的多种N-糖链结构的详细分析，这将有助于后续利用这些细胞来产生异源糖蛋白。(C)2005年威利期刊公司。
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 similar to 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 (similar to 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. (c) 2005 Wiley Periodicals, Inc.