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JAC Advance Access published online on April 5, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm036
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily

Gerda Szakonyi1, Dong Leng1, Pikyee Ma1, Kim E. Bettaney1, Massoud Saidijam1, Alison Ward1, Saeid Zibaei1, Alastair T. Gardiner2, Richard J. Cogdell2, Patrick Butaye3, Anne-Brit Kolsto4, John O'Reilly1, Ryan J. Hope1, Nicholas G. Rutherford1, Christopher J. Hoyle1 and Peter J. F. Henderson1,*

1 Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, Institute for Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK 2 Division of Biochemistry and Molecular Biology, Glasgow Biomedical Research Centre, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8TA, Scotland, UK 3 Department of Bacteriology and Immunology, Veterinary and Agrochemical Research Centre, VAR-CODA-CERVA, Groeselenberg 99, B-1180 Ukkel, Belgium 4 Department of Pharmaceutical Biosciences, University of Oslo, PO BOX 1068 Blindern, 0316 Oslo, Norway

Received 12 July 2006; returned 3 August 2006; revised 23 January 2007; accepted 26 January 2007

* Corresponding author. Tel: +44-113-343-3175; Fax: +44-113-1407; E-mail: p.j.f.henderson{at}leeds.ac.uk

A genomic strategy for the overexpression of bacterial multidrug and antibiotic resistance membrane efflux proteins in Escherichia coli is described. Expression is amplified so that the encoded proteins from a range of Gram-positive and Gram-negative bacteria comprise 5% to 35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGS(His)6-tag or a Strep-tag at the C terminus. These tags facilitate the purification of the overexpressed proteins in milligram quantities for structural studies. The strategy is illustrated for the bicyclomycin resistance efflux protein, Bcr, of E. coli.

Key Words: membrane transport proteins , antibiotic resistance , bicyclomycin , multidrugs , pathogens , Helicobacter pylori , Brucella spp. , Escherichia coli


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