Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.712643
Title: Structural studies of the endotoxin sensing protein Factor C
Author: McClymont, Karen
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2017
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Abstract:
Human exposure to Gram-negative bacterial endotoxin can result in life-threatening conditions including sepsis and toxic shock syndrome. Given the importance of avoiding exposure to endotoxin, a sensitive test is required to ensure that medical devices and injectable medicines are not contaminated. Limulus amebocyte lysate (LAL), produced from the blood of horseshoe crabs, contains an endotoxin-sensing protein, Factor C, that initiates a coagulation cascade. This lysate is used to test for contamination of medical products, with formation of a clot indicating the presence of endotoxin in the sample. Alternative methods to the LAL test have been explored to overcome problems with the test. These include the detrimental effects that harvesting has on the wild population of horseshoe crabs. More recently, tests relying on the direct detection of enzymatically active Factor C have been introduced, some of which utilize Factor C produced recombinantly. Despite the importance of Factor C to the health industries, no detailed explanation of its mechanism of endotoxin recognition has been published. The aim of this project was therefore to develop a better understanding of how Factor C is activated by endotoxin binding and to identify conformational changes that take place as a result. Two main strategies were employed for structural characterisation and functional analysis of Factor C. First, fragments of Factor C protein were produced recombinantly in Escherichia coli to determine the structures of the individual domains and identify the endotoxin binding region more precisely. Second, expression of the whole Factor C protein in eukaryotic expression systems was attempted to produce material for experiments that would shed light on any overall conformational change that occurs. Factor C fragments were successfully expressed in E. coli and purified. Circular dichroism spectroscopy identified that the majority of these fragments were folded, and changes in the spectra of some fragments in the presence of lipopolysaccharide identified potential binding sites. A preliminary structure of the first pair of complement control domains has been determined by nuclear magnetic resonance spectroscopy. Comparison of this structure with other LPS binding proteins will pave the way for future work to characterise the endotoxin binding site of Factor C.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.712643  DOI: Not available
Keywords: QD Chemistry
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