title: Expression, purification and properties of multidrug efflux proteins.
abstract: A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H(+) antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1-25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni(2+)-nitrilotriacetate-affinity column chromography, yielding 5-25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of alpha-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with (13)C or (15)N have been synthesized to implement solid-state NMR studies of the ligand-binding site.
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K. p.j.f.henderson@leeds.ac.uk
authors.forename: P J
authors.initials: PJ
authors.lastname: Henderson
authors.forename: C K
authors.initials: CK
authors.lastname: Hoyle
authors.forename: A
authors.initials: A
authors.lastname: Ward
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism; D001562:Benzimidazoles / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D017017:Methanococcus / Q000378:metabolism; D008954:Models, Biological; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013211:Staphylococcus aureus / Q000378:metabolism; D013997:Time Factors
chemical_list: D001562:Benzimidazoles; D002352:Carrier Proteins; D005456:Fluorescent Dyes; C017807:bisbenzimide ethoxide trihydrochloride