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Title: Structure, function and mechanism of beta-barrel protein assembly in Campylobacter jejuni
Author: Hardcastle, M. J.
ISNI:       0000 0004 7233 929X
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2018
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Campylobacter jejuni is one of the world’s leading causes of food-mediated bacterial infection, affecting millions of people every year. BamA, a highly conserved, essential membrane protein found in C. jejuni and other Gram-negative bacteria is responsible for folding a number of essential β-barrel outer membrane proteins making it vital for survival in this pathogenic organism. Along with essential roles in basic physiological functions, such as nutrient import and export, outer membrane proteins are also found in a number of multidrug resistance and virulence mechanisms in pathogenic organisms, making them and BamA popular targets for therapeutic intervention. BamA is well characterised in model organisms, however the conversed mechanism which facilitates assembly in different species remains unclear. In this work, the biogenesis of β-barrel proteins is investigated in C. jeuni NCTC-11168. A recombinant expression system is developed and used to express recombinant CjBamA (Cj0129) in an E. coli BL21 host, followed by subsequent purification and biophysical characterisation. A pelB leader sequence is used to target the recombinant protein for outer membrane localisation, with an extended 10x-polyhistidine tag and purified by immobilised metal affinity chromatography. Secondary structure analysis is performed using synchrotron radiation circular dichroism confirming the presence of a dominant β-sheet fraction. Furthermore, bioinformatics tools are used to investigate the structure of BamA homology models and explore the structural relationships between BamA and a previously unidentified accessory BamD protein found in the Campylobacter genus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP501 Animal biochemistry