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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP Binding Cassette Transporter, Subfamily B / biosynthesis
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ATP Binding Cassette Transporter, Subfamily B / genetics*
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ATP Binding Cassette Transporter, Subfamily B / isolation & purification*
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Anti-Bacterial Agents / pharmacology*
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Bridged Bicyclo Compounds, Heterocyclic / pharmacology
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Circular Dichroism
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Cloning, Molecular
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Crystallization
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Drug Resistance, Bacterial / drug effects
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Drug Resistance, Bacterial / genetics
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Amplification
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Genes, Bacterial / genetics
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Histidine / metabolism
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Plasmids / genetics
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Spectrophotometry, Ultraviolet
Substances
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ATP Binding Cassette Transporter, Subfamily B
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Anti-Bacterial Agents
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Bridged Bicyclo Compounds, Heterocyclic
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Histidine
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bicozamycin