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

    Author
    Wolff, M.W.; Murhammer, D.W.; Linhardt, Robert J.
    ORCID
    https://orcid.org/0000-0003-2219-5833
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    Other Contributors
    Date Issued
    1999
    Subject
    Biology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
    Degree
    Terms of Use
    In Copyright : this Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). https://rightsstatements.org/page/InC/1.0/;
    Full Citation
    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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    URI
    https://doi.org/10.1080/10826069908544689; https://hdl.handle.net/20.500.13015/5883
    Abstract
    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.;
    Description
    Preparative Biochemistry and Biotechnology, 29, 1-21; Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
    Department
    The Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
    Relationships
    The Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; https://harc.rpi.edu/;
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    https://login.libproxy.rpi.edu/login?url=https://doi.org/10.1080/10826069908544689;
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