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|Title:||Purification and 3-D structural analysis of oligomeric human multidrug transporter ABCG2|
|Citation:||Structure, 2006; 14(11):1623-1632|
|Christopher A. McDevitt, Richard F. Collins, Michael Conway, Szabolcs Modok, Janet Storm, Ian D. Kerr, Robert C. Ford and Richard Callaghan|
|Abstract:||ABCG2 is a multidrug efflux pump associated with resistance of cancer cells to a plethora of unrelated drugs. ABCG2 is a “half-transporter,” and previous studies have indicated that it forms homodimers and higher oligomeric species. In this manuscript, electron microscopic structural analysis directly addressed this issue. An N-terminal hexahistidine-tagged ABCG2R482G isoform was expressed to high levels in insect cells. An extensive detergent screen was employed to effect extraction of ABCG2R482G from membranes and identified only the fos-choline detergents as efficient. Soluble protein was purified to >95% homogeneity by a three-step procedure while retaining the ability to bind substrates. Cryonegative stain electron microscopy of purified ABCG2R482G provided 3D structural data at a resolution of 18 Å. Single-particle analysis revealed that the complex forms a tetrameric complex (180 Å in diameter × 140 Å high) with an aqueous central region. We interpret the tetrameric structure as comprising four homodimeric ABCG2R482G complexes.|
|Appears in Collections:||Molecular and Biomedical Science publications|
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