A major challenge in cancer treatment is acquired or intrinsic multidrug resistance (MDR) to chemotherapeutics. A notorious mediator of MDR is P-glycoprotein (P-gp, ABCB1), product of the human MDR1 gene, which actively effluxes cytotoxic drugs from cancer cells, resulting in sub-therapeutic intracellular concentrations. Understanding how P-gp interacts with drugs has been severely limited by the lack of high-resolution structures of P-gp. Although numerous efforts to obtain an X-ray crystal structure of P-gp have been attempted, human P-gp has never been crystallized. However, mouse P-gp (87% homologous to human P-gp) has been crystallized, and several structures of mouse P-gp have been recently reported. Despite a high degree of homology, it is currently unknown why mouse P-gp can be crystallized while human P-gp cannot. The studies presented in this thesis describe the creation of novel chimeras of mouse and human P-gp as an approach to investigate whether specific protein domains are responsible for differences in the ability to form crystals between mouse and human P-gp. A range of chimeras, created by protein domain swapping, were expressed in mammalian cells and all were found to retain MDR transport function demonstrating that P-gp can tolerate major structural changes. High-level expression of all chimeras was achieved by baculovirus-mediated heterologous protein expression. Chimeric proteins were purified by a multi-step process including immobilized metal affinity chromatography and size exclusion chromatography. Crystallization screening obtained protein crystals for two of the chimeras, indicating the approach adopted is a successful strategy, and an advance along the path towards a high-resolution structure of human P-gp.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647697 |
| Date | January 2015 |
| Creators | Pluchino, Kristen Marie |
| Contributors | Gill, Deborah |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:4f5c9dcc-401c-4123-9b2f-c5d17e0d7bc2 |
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