Our current studies are focused on the implementation of a biomimetic approach to create solid-supported membranes interfaced with nanostructures. Nanoporous membrane structures have been employed as a new substrate used to functionally immobilize the members of the ATP binding cassette (ABC) transporter superfamily. These assemblies are being used to build highly miniaturized in vitro models of multidrug resistance for high-throughput screening and drug discovery.

The drug efflux pump PDR5-GFP has been isolated from S. cerevisiae clones from a commercial library (InVitrogen) using affinity chromatography. We have constructed anti-GFP Sepharose beads via CNBr coupling with high binding capacities as support for large-scale affinity purification and have isolated PDR5-GFP at 0.1 mg scale. Furthermore, we've also begun to biosynthesize the human multidrug resistance transporter ABCG type 2 using Cell-Free Protein Synthesis (CFPS). Confocal fluorescence microscopy was utilized to analyze supported membranes constructed on 5 micron silica beads and in nanoporous anodic aluminum oxide (AAO) membrane filters with 200 nm and 100 nm pore diameters. We have begun to study the mobility and localization of transporters in supported membrane systems and have initiated fluidity studies of the supported membranes using fluorescence recovery after photobleaching (FRAP). Confinement of transporter assays to nanopores could lead to a highly miniaturized method for the analysis of ABC transporters that utilize miniscule sample amounts such as obtained from tissue biopsies.

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