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Title: Utilisation of amber suppression/non-natural amino acid technology for protein engineering and cellular control
Author: Morris, Josephine Lydia
ISNI:       0000 0004 2742 6941
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2013
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The amber suppression technology is an intracellular methodology that allows position specific incorporation of a specific non-natural amino acid (NAA) into proteins using imported NAA-specific machinery during protein translation. The method has been utilised to incorporate over 50 NAAs into proteins (e.g. those that confer unique reactivity (and allow subsequent conjugation of additional factors), installation of post-translational modification mimics, modulation of protein function and those that aid in structural determination). Therefore, the aim of the work presented within this Thesis was to explore the use of this technology in novel applications; namely the installation and study of a reactive moiety within a defined environment and the creation of a ‘biological switch’ to control the production of the protein and subsequently a cellular phenotype. It was demonstrated that incorporation of the NAA, azidophenylalanine, within the hydrophobic pocket of T4 Lysozyme (T4LazF) could provide a protein scaffold to stabilise, shield and thereby allow exploration of the chemical reactivity of the photoreactive aryl azide moiety using various spectroscopic techniques. Specifically, electron paramagnetic spectroscopy of irradiated T4LazF demonstrated that the singlet phenyl nitrene species had been captured. To create a ‘biological switch’ to control the production of a protein and the subsequent cellular phenotype, the duality of the amber suppression method was the basis for the novel application rather than the chemical reactivity of the NAA. E. coli and mammalian cell motility readout systems were successfully created using flagellin (non-flagellate) and Rac1 (GTPase protein involved in lamellipodia production) knockout cell lines respectively in conjunction with specially created plasmid constructs. However, complexities regarding the implementation of amber suppression in order to control this motility via the presence/absence of NAA limited the functionality of these readout systems. The work presented has advanced the field of amber suppression and NAA technology by demonstrating that generating reactive intermediates derived from NAAs within a defined chemical environment of a protein provides a novel technique to generate and study highly reactive intermediates. In addition, it has been shown that the amber suppression technology has potential to act as a biological switch to control cellular responses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QD Chemistry