Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli

Protein Expr Purif. 2012 Apr;82(2):396-401. doi: 10.1016/j.pep.2012.02.005. Epub 2012 Feb 20.


F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus / enzymology*
  • Bacterial Proteins / biosynthesis
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / isolation & purification
  • Culture Media
  • Culture Techniques
  • Dimyristoylphosphatidylcholine / chemistry*
  • Enzyme Assays
  • Escherichia coli
  • Lipid Bilayers / chemistry*
  • Membranes, Artificial
  • Mitochondrial Proton-Translocating ATPases / biosynthesis
  • Mitochondrial Proton-Translocating ATPases / chemistry*
  • Mitochondrial Proton-Translocating ATPases / isolation & purification
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification


  • Bacterial Proteins
  • Culture Media
  • Lipid Bilayers
  • Membranes, Artificial
  • Recombinant Proteins
  • mitochondrial ATPase subunit c
  • Mitochondrial Proton-Translocating ATPases
  • Dimyristoylphosphatidylcholine