Expression and Purification of the High-Affinity Phosphate Transporter of Saccharomyces Cerevisiae

Eur J Biochem. 1995 Jan 15;227(1-2):566-72. doi: 10.1111/j.1432-1033.1995.tb20426.x.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / genetics
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • DNA, Recombinant
  • Escherichia coli / genetics
  • Membrane Proteins / genetics*
  • Membrane Proteins / isolation & purification
  • Membrane Transport Proteins / genetics*
  • Membrane Transport Proteins / isolation & purification
  • Molecular Sequence Data
  • Phosphate Transport Proteins*
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / genetics
  • Saccharomyces cerevisiae / genetics*

Substances

  • DNA, Recombinant
  • Membrane Proteins
  • Membrane Transport Proteins
  • Phosphate Transport Proteins
  • Recombinant Fusion Proteins
  • phosphate permease
  • Alkaline Phosphatase