Release and Preparation of Intact and Unreduced N-linkedOligosaccharides from Sf-9 Insect Cells

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Wolff, M.W.
Murhammer, D.W.
Linhardt, Robert J.
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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Glycosylation, the addition of carbohydrates to a peptide backbone, is the most extensive cotranslational and posttranslational modification made to proteins by eukaryotic cells. The glycosylation profile of a recombinant glycoprotein can significantly affect its biological activity, which is particularly important when being used in human therapeutic applications. Therefore, defining glycan structures to ensure consistency of recombinant glycoproteins among different batches is critical. In this study we describe a method to prepare N-linked glycans derived from insect cell glycoproteins for structural analysis by capillary electrophoresis. Briefly, glycoproteins obtained from uninfected Spodoptera frugiperda Sf-9 insect cells were precipitated with ammonium sulfate and the glycans were chemically cleaved by hydrazinolysis. Following the regeneration of the glycan reducing terminal residue and the removal of contaminating proteins and peptides, the glycans were fluorescently labeled by reductive amination. Fluorescent labeling greatly enhanced the detection limit of the glycan structures determined by capillary electrophoresis. Five major glycan structures were found that migrated between tetra-mannosylated hexasaccharide and nonamannosylated undecasaccharide standards. Upon alpha-mannosidase digestion the number of glycan structures was reduced to two major structures with shorter migration times than the undigested glycans. None of the glycans were susceptible to hexosaminidase or galactosidase treatment. These results are consistent with the majority of previous results demonstrating hypermannosylated glycan structures in Sf-9 insect cells.
Preparative Biochemistry and Biotechnology, 29, 1-21
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Release and Preparation of Intact and Unreduced N-linkedOligosaccharides from Sf-9 Insect Cells, M. W. Wolff, D. W. Murhammer,R.J. Linhardt, Preparative Biochemistry and Biotechnology, 29,1-21, 1999.
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