RESUMEN
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.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/aislamiento & purificación , Antibacterianos/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Dicroismo Circular , Clonación Molecular , Cristalización , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Amplificación de Genes , Genes Bacterianos/genética , Histidina/metabolismo , Plásmidos/genética , Espectrofotometría UltravioletaRESUMEN
Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.
Asunto(s)
Bacterias/metabolismo , Farmacorresistencia Bacteriana , Proteínas de Transporte de Membrana/metabolismo , Secuencia de Aminoácidos , Bacterias/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de ProteínaRESUMEN
A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative alpha-ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C-terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Dicroismo Circular , Clonación Molecular , ADN Bacteriano/genética , ADN Recombinante/genética , Escherichia coli/genética , Genes Bacterianos , Vectores Genéticos , Helicobacter pylori/genética , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
Helicobacter pylori is a common pathogen of humans, which predisposes individuals to gastric inflammation and a variety of diseases including gastric cancer. Sequencing of the organism's genome reveals an extensive portfolio of predicted membrane transport proteins, although few of the proteins encoded by these genes have yet been isolated. We describe here the cloning and expression in Escherichia coli of the H. pylori gene hp1181 encoding a putative multidrug resistance membrane transport protein. Substantial overexpression was accomplished, and the protein was tagged with RGS(His)(6) at the C-terminus, which enabled its purification in mg quantities. Identification of the full-length protein was achieved by N-terminal amino acid sequencing and Western blotting using an antibody to the RGS(His)(6) epitope. The retention of structural integrity and occurrence of predicted alpha-helix in the protein were verified by circular dichroism spectroscopy.