Expression, purification and properties of multidrug efflux proteins

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. co li. 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 plas mid pTTQ18; an oligonucleotide encoding six histidine residues was added, i n frame, to the C-terminus to facilitate purification. Growth conditions we re 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 assa ys of antibiotic resistance in vivo and/or assays of energized transport ac tivity in vitro with synthetic substrates. Proteins were solubilized in dod ecylmaltoside and purified to more than 90%, homogeneity with Ni2+-nitrilot riacetate-affinity column chromography, yielding 5-25 mg per 25 litres of o riginal culture. All the transport proteins migrated anomalously in SDS/PAC E 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. Exami nation of the secondary structure of detergent-solubiIized proteins by CD o r Fourier-transform infrared spectroscopy following purification indicated a high content of alpha-helix (more than 75 %). Matrix-assisted laser desor ption ionization MS confirmed the high degree of purity and the true molecu lar mass. The formation of three-dimensional crystals is being attempted bu t crystals have yet to be grown that diffract X-rays. The growth of two-dim ensional protein arrays has been more successful, with diffraction of elect rons at low resolution. Proteins have been fused to green fluorescent prote in or maltose-binding protein to facilitate these structural analyses. In a ddition, ligands for efflux proteins labelled with C-13 Or N-15 have been s ynthesized to implement solid-state NMR studies of the ligand-binding site.