Thomas, Daniel Stephen
2019.
Modification of human thyroid peroxidase for structural studies.
PhD Thesis,
Cardiff University.
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Abstract
Thyroid peroxidase (TPO) is an enzyme found on the cell surface of thyrocyte cells which is involved in the production of thyroid hormones. TPO is one of three major autoantigens in autoimmune thyroid disease (AITD). Despite investigations by several groups, the three-dimensional structure of the TPO protein is unknown. Solving the structure of TPO would further the understanding of AITD and produce useful data for improving the diagnosis and treatment of the disease. The focus of this work was to use information from the structures of related peroxidase enzymes to design modified TPO proteins which would be suitable for X-ray crystallography. Five modified proteins were designed and expressed in an insect cell line. Of these five proteins, four were successfully purified and concentrated to the high levels required for crystallographic studies. These proteins were characterised to show that they retained key features of the native protein such as autoantibody binding and enzymatic activity. The four proteins were tested in sparse matrix crystal screens. Two of the proteins produced large, single crystals suitable for X-ray diffraction experiments. The diffraction data obtained from these crystals had a maximum resolution of 5.2 Å, an improvement on previous work but insufficient to produce a reliable structure. Further experiments were carried out but the diffraction resolution could not be improved. These involved varying the crystallization conditions, modifying the protein by deglycosylation and using ligands to stabilise the protein. Additional work focused on furthering the understanding of autoantibody binding to TPO. Using an epitope excision technique not previously applied to TPO, it was demonstrated that an anti-TPO monoclonal antibody bound to a region close to or covering one of two immunodominant regions of the protein. These results are in good agreement with data from the current literature.
Item Type: | Thesis (PhD) |
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Date Type: | Completion |
Status: | Unpublished |
Schools: | Chemistry |
Date of First Compliant Deposit: | 3 April 2020 |
Last Modified: | 12 Apr 2021 10:12 |
URI: | https://orca.cardiff.ac.uk/id/eprint/130769 |
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