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Characterisation of Truncated Mutant Rhodopsin and its Involvement in the Pathogenesis of Retinitis Pigmentosa

Hargreaves, Hannah N M (2020) Characterisation of Truncated Mutant Rhodopsin and its Involvement in the Pathogenesis of Retinitis Pigmentosa. Masters thesis, University of Essex.

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Abstract

Autosomal dominant retinitis pigmentosa (ADRP) is the most common genetic disorders which cause visual degradation, and blindness. 20-25% of cases are caused by mutations in the rhodopsin gene. The mutations located in the N-terminus of rhodopsin produce severely misfolded protein, which has been shown to cleave at the N-terminus removing the glycosylation sites(Tam & Moritz, 2007),(Krebs, et al., 2010). We aimed to further understand the pathology of retinitis pigmentosa by separating full rhodopsin from the truncated rhodopsin of the mutant, and we constructed rhodopsin N-terminus ADRP mutations: T4K, P23A/H and Q28H. Wild Type and mutant rhodopsin were purified using 1D4-sepharose separation. Our results supported papers indicating P23A and T4K show lowest degrees of misfolding. Assuming the glycosylation sites are removed we attempted to isolate the truncated species of the ADRP mutant by Con A-Sepharose which binds to the glycan moieties extended from the glycosylation sites N2 and N15(De Grip, W. J.,1982).We isolated purified ROS rhodopsin and full P23A rhodopsin. We failed to isolate the P23A truncated species, and poor binding was apparent from full rhodopsin in the wash and flow through after binding. Meaning the truncated species if present would not have been isolated. Further alterations are needed to make the Con A-sepharose separation successful.

Item Type: Thesis (Masters)
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Hannah Hargreaves
Date Deposited: 25 Jul 2020 09:37
Last Modified: 25 Jul 2020 09:37
URI: http://repository.essex.ac.uk/id/eprint/28151

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