Expression and purification of the high-affinity phosphate transporter of Saccharomyces cerevisiae.
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.
Berhe
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Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, University of Stockholm, Sweden.
Fristedt
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U
Persson
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BL
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D028061:Phosphate Transport Proteins; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004274:DNA, Recombinant; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D028061:Phosphate Transport Proteins; D011993:Recombinant Fusion Proteins; C055831:phosphate permease; D000469:Alkaline Phosphatase