The breast cancer resistance protein (BCRP) is an ATP Binding Cassette (ABC) transporter responsible for the export of many toxins across cellular membranes. Overexpression of these ABC transporters is often correlated to multidrug resistances in cancer and decrease in the success of chemotherapy. We have performed high‐throughput in silico ligand docking studies to find drug‐like compounds that are predicted to inhibit the function of BCRP and the related transporter, P‐glycoprotein. A number of drug like molecules were identified that reversed multidrug resistance of a BCRP‐overexpressing breast cancer cell line generated in our lab. Biochemical and biophysical analyses of these inhibitors are needed to evaluate the inhibition mechanism of these drug‐like compounds. For that purpose, relatively large amounts of the purified protein are required. We previously generated a gene for human BCRP that was codon optimized for expression in the yeast, Pichia Pastoris, and cloned into the new expression system PichiaPinkTM. BCRP purification proved to be challenging as reported previously by others. Optimization of growth and BCRP expression in the PichiaPinkTM cells revealed highest cell density and satisfactory BCRP expression after methanol induction when the cells were fed with their carbon source, methanol, more frequently than originally described. Protein purification techniques including cell breakage and solubilization conditions were optimized to achieve a consistent 60% membrane solubilization. Here we report our attempts to purify BCRP into detergent mixed micelles using Ni‐NTA affinity and ion exchange chromatography. Our attempts to assemble BCRP into native‐like membrane nanodiscs to achieve maximum ATP‐hydrolysis activity are also reported. Obtaining BCRP in a near native like environment with high biological activity will be necessary for the assessment of efficiency as well as the mechanism of BCRP inhibition of our newly found drug‐like compounds.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors wish to thank Ms. Lynn McBee and New England Biolabs for their generous support.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.