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Investigation of CHO cell surface glycosylation using lectin probes.
Cawley, Jonathan
Chinese hamster ovary (CHO) cells are extensively used for the production of therapeutic proteins which frequently undergo post-translational modifications (PTM), including glycosylation. This process can alter the function, stability, immunogenicity and efficacy of the glycoprotein. There are many culture conditions that may affect glycosylation. The CHO glycocalyx, carbohydrate coat surrounding the cell membrane, may be informative to cell health and the glycosylation state of the product. It is therefore of great interest to understand how the CHO glycocalyx changes in different culture conditions. Fluorescent microscopy and flow cytometry were used to glycoanalyse CHO DP-12 cells. Novel recombinant lectins were mutated for optimum fluorescent labelling and purified using immobilized metal affinity chromatography (IMAC). Lectins from non-plant sources are perfect glycan probes as they are generally non-toxic but also superior to other binding proteins such as antibodies whose specificities for glycans are ill-defined. In this study a eukaryotic lectin, Agrocybe aegerita lectin 2 (AAL-2), and prokaryotic lectins from Pseudomonas aeruginosa (LecA) and Escherichia coli (GafD1-178) were modified, recombinantly produced, purified and characterised. LecA was modified to optimise its labelling (biotinylation) for subsequent use as a fluorescently labelled lectin probe. CHO DP-12 cells underwent various culture media treatments, e.g. spent medium, 0 mM L-glutamine, 10 mM NH4Cl or 3 mM NaBu, and the glycocalyx was investigated with a panel of lectins. Lectin binding changed significantly after culture conditions were altered. MAL II binding (sialic acid) decreased 43.79 % following spent medium treatment. A simultaneous 229 % increase in recombinant LecA, a galactophilic lectin, binding was observed which dropped to 32.24 % when the cells were reseeded in fresh medium. Recombinant IgG1 was also purified from treated CHO DP-12 cultures and glycoanalysed by an optimised enzyme-linked lectin assay (ELLA). The recombinant lectins produced were highly appropriate for investigating the glycocalyx of live CHO DP-12 cells.
Keyword(s): Biotechnology; Biochemistry; Molecular biology; Lectin; Glycosylation
Publication Date:
2017
Type: Doctoral thesis
Peer-Reviewed: No
Language(s): English
Institution: Dublin City University
Citation(s): Cawley, Jonathan (2017) Investigation of CHO cell surface glycosylation using lectin probes. PhD thesis, Dublin City University.
Publisher(s): Dublin City University. School of Biotechnology; Dublin City University. Irish Separation Science Cluster (ISSC); Dublin City University. National Centre for Sensor Research (NCSR)
File Format(s): application/pdf
Supervisor(s): O'Connor, Brendan
Walls, Dermot
Related Link(s): http://doras.dcu.ie/21960/1/J_Cawley_-_Thesis.pdf
First Indexed: 2017-11-30 06:05:46 Last Updated: 2018-05-15 06:05:03