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Title: Over-expression, purification and site-directed mutagenesis of factor C and the influence of redox
Author: Hezwani, Mohammed Haythem
Awarding Body: University of the West of England, Bristol
Current Institution: University of the West of England, Bristol
Date of Award: 2013
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Introduction & Aims: Not only is sepsis a devastating clinical syndrome it has immense direct and indirect costs to the NHS. Sepsis is caused by bacterial molecules, the most common, lipopolysaccharide (LPS). The current diagnostic assay utilises Factor C (FC) a protein that initiates the coagulation cascade in horseshoe crabs, which activates in the presence of minute amounts of LPS forming the basis of the Pyrogene® assay. However, activation of FC in this assay requires an hour to complete, a reaction that occurs within seconds in its native environment, suggesting that key chaperones are absent from the commercial assay. FC is a thiol rich protein and our hypothesis is that changes in the redox environment together with a deeper understanding of potential unidentified chaperones will improve LPS activation, ultimately reducing the assay time. Methods: FC was over-expressed and purified using Gateway® cloning system and a series of purification techniques. Western blot analysis and the FC assay assessed the efficiency of purified FC under various redox conditions. Finally, using targeted affinity proteomics key proteins involved in LPS binding were identified. Results & Implications: FC was successfully over-epxressed and partially purified using its GST-tag. Further optimisation of FC purification should be considered. This formed the template for the generation of 6 CXXC mutants, (3X N-terrninal and 3X double thiol mutants) that showed altered FC activity. The activity of the N-terminal mutants was reduced by up to 20% (G323C), suggesting that the N-terminal cysteine residue is required for the function of FC. However, double CXXC mutants improved activity by 20% (G323C-G332C) indicating that the protein can correctly fold resulting in more factorable LPS binding conditions. The importance of the redox environment is further demonstrated when the activity of FC is lost when incubated with the reducing agent DD, however, oxidising agents improved the activity. These findings confirm that disulphide bonding is key in regulating FC activity. Finally, targeted affinity proteomics identified Tachylectin-2, Hemagglutinin/Amebocyte aggregation factor (18K-LAF), which suggests that they may play similar roles to LBP and CD14 signalling. These proteins can be further investigated by including them in current LPS detection assays to assess if they improve on Fe activity and reactions times. More interestingly, the identification of Coagulogen, that has not previously been implicated in LPS binding, has led to the hypothesis that it these proteins may interact and form a metabolon, which may give an insight as to how the horseshoe crab can complete the cascade in 90 seconds.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available