The innate immune system provides the body’s first line of defense against pathogenic challenge through pathogen recognition and initiation of the immune response. Among the various cellular mechanisms of pathogen recognition in mammals, Toll-like receptor 3 (TLR3) recognizes viral dsRNA. Stimulation of TLR3 signaling pathway leads to transcription of pro-inflammatory cytokines and type-1 Interferons. Suppressor of IKKε (SIKE) interacts with two kinases in the signaling pathway, IKKε and TANK binding kinase 1 (TBK1), inhibiting the transcription of type I interferons. Recently, the Bell Laboratory discovered that SIKE blocks TBK1-mediated activation of type I interferons by acting as a high affinity, alternative substrate of TBK1.
To further characterize SIKE’s function within the antiviral response, this study focused on defining the overall SIKE structure. Using recombinant protein expressed from E. coli and purified via immobilized metal affinity chromatography, SIKE crystals were obtained from a sample concentrated to 15 mg/ml under several crystallization conditions. Yet, reproducing these results has been difficult. In this study, we have modified the purification scheme to remove an E. coli contaminant, SlyD. Purification under denaturing conditions, removal of soluble proteins, incorporation of ion exchange and different IMAC (immobilized metal ion affinity chromatography) resins has been tested. For each scheme, size exclusion chromatography and SDS-PAGE/Coomassie/silver stain were used to assess purity. Crystallization trials for samples from each purification scheme were completed. In addition to crystallization trials, hydrogen-deuterium exchange (HDX) was investigated, accompanied with pepsin digests, in order to further characterize the dynamic structure of SIKE.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-4695 |
| Date | 01 January 2014 |
| Creators | McKinley, Sean W |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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