title: A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily.
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.
authors.affiliation: Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, Institute for Membrane and Systems Biology, University of Leeds, Leeds, UK.
authors.forename: Gerda
authors.initials: G
authors.lastname: Szakonyi
authors.forename: Dong
authors.initials: D
authors.lastname: Leng
authors.forename: Pikyee
authors.initials: P
authors.lastname: Ma
authors.forename: Kim E
authors.initials: KE
authors.lastname: Bettaney
authors.forename: Massoud
authors.initials: M
authors.lastname: Saidijam
authors.forename: Alison
authors.initials: A
authors.lastname: Ward
authors.forename: Saeid
authors.initials: S
authors.lastname: Zibaei
authors.forename: Alastair T
authors.initials: AT
authors.lastname: Gardiner
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Cogdell
authors.forename: Patrick
authors.initials: P
authors.lastname: Butaye
authors.forename: Anne-Brit
authors.initials: AB
authors.lastname: Kolsto
authors.forename: John
authors.initials: J
authors.lastname: O'reilly
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Hope
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Rutherford
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hoyle
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D005798:Genes, Bacterial / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000900:Anti-Bacterial Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D006639:Histidine; C024504:bicozamycin