The research described here focuses on the molecular variations, clinical significance and structural interactions of the innate immune protein, C-reactive protein (CRP). Monomeric CRP (mCRP) was produced through urea-induced dissociation, at an optimum concentration of 3M urea over a 10-week period. Dissociated samples were purified via size exclusion chromatography and confirmation was provided via Western Blot analysis. In contrast with previous published works, this mCRP retained the ability to reversibly bind to phosphocholine. Furthermore, the binding of mCRP with cell wall polysaccharide does not result in the generic precipitation trend as that seen with pentameric CRP (pCRP). Human serum samples with raised CRP levels ( > 100mg/L) were analysed (n =40) to determine whether circulating mCRP could be detected in vivo. All 40 samples tested positive for pCRP via Western Blot and ELISA analysis, with further quantification via the UV Spectrophotometer supporting the calculated pCRP levels. Monomeric CRP was identified in all the 40 patient samples tested, with an average value recorded of 0.88mg/L (SE = ±0.142). Informed by the results from in vitro mCRP studies, the mCRP displayed a molecular weight of approximately 25kDa based on size exclusion chromatography and Western Blot analysis and demonstrated an ability to reversibly bind phosphocholine. To our knowledge, we are the first to successfully purify and identify a monomeric form of CRP from human serum through these procedures. Crystallisation trials of in vitro produced mCRP and the complex of pCRP with C1qGHR and CWPS did not result in the growth of any protein crystals of sufficient diffraction quality. One crystal from the complex crystallisation trials diffracted to 9Å with follow up conditions currently being pursued. Furthermore, additional crystallisation trials from both experiments have generated potential conditions which may be refined in future research to produce high-quality diffraction grade crystals.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:718476 |
| Date | January 2017 |
| Creators | Williams, Robert D. |
| Publisher | Keele University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.keele.ac.uk/3528/ |
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