Expression and purification of the high-affinity phosphate transporter of Saccharomyces cerevisiae

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.