Expression of ABC Transporters in Pichia pastoris(RMI)

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DESCRIPTION (provided by applicant): The ATP-binding cassette (ABC) family of membrane transporters has over 40 members in humans. These proteins fulfill key physiological roles as they transport a number of important physiological substrates such as lipids, peptides, ions, and xenobiotics in different organs and cell types. The important role of ABC transporters is highlighted by the fact that inactivating mutations in several of these transporters cause debilitating diseases in humans, including cystic fibrosis, immunodeficiency, blindness, and defects in lipid and cholesterol metabolism. Also, overexpression of ABC transporters such as P-glycoproteins and MRP cause cellular resistance to drugs and xenobiotics, including drugs used for chemotherapy of many types of human cancers. Many aspects of ABC transporter function remain poorly understood, including the identity of physiological substrates, the mechanism of transport, and the nature of the defect in associated human pathologies. In addition, there is very little structural information available for these mammalian transporters. We have previously succeeded in expressing large amounts of biologically active members of the ABCB and ABCC family in the yeast Pichia pastoris and have been able to isolate large amounts of pure, biologically active ABCB proteins in quantities sufficient for structural studies by X-ray crystallography. We propose to extend these studies and systematically express all ABC transporters of relevance for human disease in P. pastoris. The specific aims of this proposal include: identifying clones expressing high amounts of the proteins, characterizing the functional status of these proteins, and purifying large amounts of individual proteins for biochemical and structural studies. Importantly, we propose an incremental approach where all useful reagents will be made available to the scientific community as they become available and are validated in our lab. The experimental burden of purifying and reconstituting these transporters would be shared by other labs with a special interest and previous experience with a specific transporter(s).