Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Bacterial Proteins / genetics*
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Bacterial Proteins / isolation & purification*
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Base Sequence
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Carrier Proteins / genetics
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Carrier Proteins / isolation & purification
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Circular Dichroism
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Cloning, Molecular
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DNA, Bacterial / genetics
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DNA, Recombinant / genetics
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Escherichia coli / genetics
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Genes, Bacterial
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Genetic Vectors
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Helicobacter pylori / genetics
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Membrane Proteins / genetics*
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Membrane Proteins / isolation & purification*
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Plasmids / genetics
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Solubility
Substances
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Bacterial Proteins
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Carrier Proteins
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DNA, Bacterial
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DNA, Recombinant
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Membrane Proteins
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Recombinant Proteins