THE MFS EFFLUX PROTEINS OF GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA By MASSOUD SAIDIJAM, Department of Molecular Medicine and Genetics, School of Medicine, Hamedan University of Medical Sciences, Hamedan, Iran, KIM E. BETTANEY, DONG LENG, PIKYEE MA, ZHIQIANG XU, JEFFREY G. KEEN, NICHOLAS G. RUTHERFORD, ALISON WARD AND PETER J. F. HENDERSON  , Institute for Membrane and Systems Biology, Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK, GERDA SZAKONYI, Institute of Pharmaceutical Analysis, Faculty of Pharmacy, University of Szeged, Szeged, Hungary, QINGHU REN, IAN T. PAULSEN, Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia, INGERID NES, JASMIN K. KROEGER, ANNE-BRIT KOLSTO  , Laboratory for Microbial Dynamics (LaMDa), Department of Pharmaceutical Biosciences, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway CONTENTS I. Introduction II. Cloning a Putative Efflux Protein into a Plasmid Expression Vector III. Cultivation of Host Cells Containing the Plasmid Encoding the Target Protein IV. Detection of Histidine-Tagged Efflux Proteins in E. coli Membrane Preparations V. Solubilization of the Efflux Proteins with Detergents VI. Purification of Histidine-Tagged Efflux Proteins VII. Confirmation of Integrity and Monodispersity of the Purified Protein VIII. Crystallization Trials on Purified Proteins IX. Conclusions and Discussion Acknowledgements References Advances in Enzymology and Related Areas of Molecular Biology, Volume 77 Edited by Eric J. Toone Copyright Ó 2011 John Wiley & Sons, Inc.  Corresponding authors: Peter J. F. Henderson (p.j.f.henderson@leeds.ac.uk) and Anne-Brit Kolsto (a.b.kolsto@farmasi.uio.no) 147