Abstract
The plasma membrane high-affinity phosphate permease of Saccharomyces cerevisiae has been overproduced as a stable membrane-bound chimeric protein in Escherichia coli. Construction of a chimera between the permease and a peptide containing 10 consecutive histidine residues allowed selective binding of the chimera to a chelating column charged with Ni2+, and elution with imidazole in a high state of purity. Approximately 5 mg purified His10-permease was obtained from 3 g (wet mass) cells. The purified phosphate permease chimera catalyzes uncoupler-sensitive phosphate transport after reconstitution into proteoliposomes.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Alkaline Phosphatase / genetics
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Amino Acid Sequence
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Base Sequence
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Cloning, Molecular
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DNA, Recombinant
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Escherichia coli / genetics
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Membrane Proteins / genetics*
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Membrane Proteins / isolation & purification
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Membrane Transport Proteins / genetics*
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Membrane Transport Proteins / isolation & purification
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Molecular Sequence Data
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Phosphate Transport Proteins*
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Protein Structure, Secondary
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Recombinant Fusion Proteins / genetics
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Saccharomyces cerevisiae / genetics*
Substances
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DNA, Recombinant
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Membrane Proteins
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Membrane Transport Proteins
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Phosphate Transport Proteins
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Recombinant Fusion Proteins
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phosphate permease
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Alkaline Phosphatase