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Investigating the effects of cyclisation on a protein tool and a biocatalyst

Hayes, Heather 2022. Investigating the effects of cyclisation on a protein tool and a biocatalyst. PhD Thesis, Cardiff University.
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As therapeutics and biocatalysts, polypeptides are attractive alternatives to traditional small molecules and catalysts, respectively. However, the applications of peptides and proteins are often limited by their instabilities towards elevated temperatures, organic solvents and/or digestive enzymes. Cyclisation by intramolecular covalent bond formation between the polypeptide chain C- and N-termini, has been demonstrated to improve stability and enhance activity. As a result, cyclisation could provide a convenient route for expanding the scope of peptide and protein application in research and industrial settings. In this thesis, the effects of cyclisation on the stabilities of two biotechnologically relevant proteins were explored. TET12 is a tetrahedral shaped artificial CCPO protein cage. Two cyclic variants of TET12 were generated using a split intein-mediated approach and a SpyTag/SpyCatcher-NTEV isopeptide bond cyclisation strategy. The thermal, chemical and proteolytic stabilities of the linear and cyclic TET12 variants were compared. Cyclisation was found to be beneficial for enhancing proteolytic resistance but thermal and chemical stability were not improved. Increased aggregation of cyclic TET12 was observed, resulting from a combination of covalent oligomerisation and protein misfolding. These results indicate that connection the polypeptide termini disrupts the TET12 folding pathway and highlights the importance of selecting an appropriate cyclisation technique. Improvements to the stability and activity of the PET degrading enzyme IsPETase, are required if it is to be utilised for large scale recycling applications. SpyTag/SpyCatcher(- NTEV) was employed to cyclise IsPETase in a range of cyclic topologies, including a cyclic monomer, cyclic dimer and catenane. The stabilities and activities of the linear and cyclic IsPETase variants were examined. In contrast to previous findings for cyclic protein topologies, the cyclic IsPETase variants did not display improved thermal stability compared to the wild type enzyme and were found to be more susceptible to trypsin digest. The dimeric variants of IsPETase exhibited enhanced PET degradation under optimal conditions and were also found to be more resistant to agitation-induced inactivation. This demonstrates the need for further investigation into the mechanisms behind protein stabilisation using cyclisation.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Chemistry
Date of First Compliant Deposit: 21 November 2022
Last Modified: 21 Nov 2022 12:41

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