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Investigation of CHO cell surface glycosylation using lectin probes.

Cawley, Jonathan (2017) Investigation of CHO cell surface glycosylation using lectin probes. PhD thesis, Dublin City University.

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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.

Item Type:Thesis (PhD)
Date of Award:November 2017
Supervisor(s):O'Connor, Brendan and Walls, Dermot
Uncontrolled Keywords:Lectin; Glycosylation
Subjects:Biological Sciences > Biotechnology
Biological Sciences > Biochemistry
Biological Sciences > Molecular biology
DCU Faculties and Centres:DCU Faculties and Schools > Faculty of Science and Health > School of Biotechnology
Research Initiatives and Centres > Irish Separation Science Cluster (ISSC)
Research Initiatives and Centres > National Centre for Sensor Research (NCSR)
Use License:This item is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 License. View License
ID Code:21960
Deposited On:13 Nov 2017 12:06 by Brendan O'Connor. Last Modified 17 Nov 2017 09:35

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