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An in vivo platform for identifying protein aggregation inhibitors

Saunders, Janet Catherine (2014) An in vivo platform for identifying protein aggregation inhibitors. PhD thesis, University of Leeds.

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Abstract

Protein aggregation is the basis of a vast array of diseases and one of the most expensive problems to overcome during production of biopharmaceuticals. The tendency of proteins to aggregate ensures that demanding purification techniques are a pre-requisite to in vitro analysis of aggregation mechanisms and screening for aggregation inhibitors. In this thesis, a powerful new system was developed to identify aggregation-prone sequences in vivo, and to screen for inhibitors of aggregation. The screen is based on a β-lactamase-tripartite fusion system, where the minimal inhibitory concentration of antibiotic, conferred by the β-lactamase enzyme, is used to evaluate the level of test protein aggregation. Using this in vivo system, the aggregation propensity of the two disease-related proteins human islet amyloid polypeptide (hIAPP) and amyloid beta peptide (Aβ) was found to be significantly higher than non-aggregating controls. Importantly, this system provides a new approach to assess aggregation-propensity without the need for purified protein. The system was used to screen small molecules for their aggregation-inhibiting properties against hIAPP. It was found that many results correlated well with the published literature on these molecules, but notably, a number did not. In vitro analysis of hIAPP aggregation in the presence of these molecules validated the results from the in vivo assay, refuting a number of published studies and confirming the power of the tripartite system for identifying aggregation inhibitors. Finally, the system was used to differentiate between an aggregating and non-aggregating human VH antibody domain, to demonstrate the application of the screen to biopharmaceuticals. The in vivo system successfully identified the aggregating test protein, wherein the addition of excipients prevented its aggregation in vivo in a titratable manner. Overall the work presented herein describes a novel, and experimentally simple, in vivo system that provides rapid and accurate analysis of protein aggregation and its inhibition.

Item Type: Thesis (PhD)
Keywords: Protein aggregation, amyloid, small molecule inhibitors, hIAPP, diabetes
Academic Units: The University of Leeds > University of Leeds Research Centres and Institutes > Astbury Centre for Structural Molecular Biology (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Identification Number/EthosID: uk.bl.ethos.638880
Depositing User: Unnamed user with username jcsaunders01
Date Deposited: 25 Feb 2015 13:47
Last Modified: 06 Oct 2016 14:42
URI: http://etheses.whiterose.ac.uk/id/eprint/7622

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