BIOT 269 |
| Human proteins often require post-translational modifications for their biological activities. Insect Drosophila melanogaster S2 cell system is a non-lytic expression system for eukaryotic foreign protein production and has a high productivity. We performed successful secreted production of human erythropoietin (hEPO) in stably transfected S2 cell system. However, this system has different N-glycosylation pattern with mammalian cell system. Thus, we tried the modification of their N-glycosylation pathway. A chemical inhibitor for hexosaminidase was applied to the culture because we guessed this enzyme is a main factor of simple N-glycosylation in Drosophila S2 cells. According to MALDI-TOF mass spectrometry or Western blot, purified S2-cells derived proteins hEPO, had a smaller molecular weight than mammalian derived native proteins. These data suggested that S2-cells derived proteins were incompletely glycosylated, so their biological activities in human body could be abnormal. We also analyzed the N-glycan itself with 2D- HPLC. From these data, we found that the proteins produced in Drosophila S2 cells have N-glycans of simple core structures. When 2-ADN was added, the culture profile was not changed and hEPO had a slightly bigger molecular weight than an original one. Its N-glycans were also analyzed by MALDI-TOF MS and the more complex structure that had a terminal N-acetylglucosamine was clearly detected. |
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Poster Session
5:30 PM-7:30 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biochemical Technology |