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 ( 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.
Kim
Yeon Kyu
YK
Department of Chemical Engineering and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Korea.
Shin
Hwa Sung
HS
Tomiya
Noboru
N
Lee
Yuan C
YC
Betenbaugh
Michael J
MJ
Cha
Hyung Joon
HJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004330:Drosophila; D004921:Erythropoietin / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011134:Polysaccharides / Q000032:analysis*; D046188:Protein Modification, Translational; D011994:Recombinant Proteins
D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin