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

Gerda Szakonyi, Dong Leng, Pikyee Ma, Kim E. Bettaney, Massoud Saidijam, Alison Ward, Saeid Zibaei, Alastair T. Gardiner, Richard J. Cogdell, Patrick Butaye, Anne Brit Kolsto, John O'Reilly, Ryan J. Hope, Nicholas G. Rutherford, Christopher J. Hoyle, Peter J.F. Henderson

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12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1265-1270
Number of pages6
JournalJournal of Antimicrobial Chemotherapy
Volume59
Issue number6
DOIs
Publication statusPublished - Jun 1 2007

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Keywords

  • Antibiotic resistance
  • Bicyclomycin
  • Brucella spp.
  • Escherichia coli
  • Helicobacter pylori
  • Membrane transport proteins
  • Multidrugs
  • Pathogens

ASJC Scopus subject areas

  • Pharmacology
  • Microbiology (medical)
  • Infectious Diseases
  • Pharmacology (medical)

Cite this

Szakonyi, G., Leng, D., Ma, P., Bettaney, K. E., Saidijam, M., Ward, A., Zibaei, S., Gardiner, A. T., Cogdell, R. J., Butaye, P., Kolsto, A. B., O'Reilly, J., Hope, R. J., Rutherford, N. G., Hoyle, C. J., & Henderson, P. J. F. (2007). A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily. Journal of Antimicrobial Chemotherapy, 59(6), 1265-1270. https://doi.org/10.1093/jac/dkm036