Generation of chimeric P-glycoprotein for functional and structural investigations

Thesis

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.

Authors: Pluchino, KM

• Publication date: 2015
• DOI: 10.5287/ora-m8awredzv
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: multidrug resistance; chimera; P-glycoprotein
• Subjects: Medical Sciences; Membrane proteins; Oncology; Crystallography; Multidrug resistance