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Title: Structural and functional analyses of key mediators of mammalian immunity
Author: Morrison, Gareth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2009
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Codon-optimized recombinant HBD2 was expressed in E.coli cells as an insoluble mutated ketosteroid isomerase (KSI) fusion protein. Efficient cyanogens bromide chemical cleavage released a soluble HBD2 peptide that upon RP-HPLC purification resulted in a highly pure reduced peptide. Oxidation using the redox partners cysteine/cystine resulted in a properly folded, functionally active BD2 as assessed by both antimicrobial killing assay and chemotactic assay using CCR6 expressing human embryonic kidney (HEK) 293 cells. Subsequent purification of HBD1 and HBD2 single disulfide bond mutants revealed that this method could be used for the future production of other ß-defensins. Single disulfide mutant HBD2 peptides were analysed to investigate the effects that removing two disulfide bonds had on ß–defensins functions. It was clearly shown that although still retaining antimicrobial activity in an in vitro assay the concentration of single disulfide mutants needed to kill an equivalent number of E.coli colonies was higher as compared to wild-type HBD2. All three structurally constraining disulfide bonds are necessary for full antimicrobial activity and chemotactic ability. CCR6 was expressed in the HEK 293 cell line and expressed with an N-terminal FLAG tag and a C-terminal decahistidine tag. CCR6 expressed on the outer surface of these mammalian cells was solubilised in mild detergents, and its purification was optimized using various chromatographic techniques. Sufficient quantity was purified to analyze the receptors glycosylation state. A gel-shift western blot assay resulted in CCR6 being the third chemokine receptor in the family to be identified as N-glycosylated. N-terminal CCR6 truncation, resulting in removal of the first 27 amino acids, was expressed in the same cell line. Although western blot analysis revealed transfection had been successful, surface expression of the mutated receptor could not be detected and as such the N-terminus has been implicated as necessary for ell surface trafficking.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available