Production and N ‐glycan analysis of secreted human erythropoietin glycoprotein in stably transfected Drosophila S2 cells

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 (∼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. Copyright © 2005 Wiley Periodicals, Inc.