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

• Published in: Protein expression and purification 2012-04, Vol.82 (2), p.396-401
• Main Authors: Yumen, Ikuko, Iwasaki, Iku, Suzuki, Toshiharu, Todokoro, Yasuto, Tanaka, Kentaro, Okada, Osamu, Fujiwara, Toshimichi, Yoshida, Masasuke, Akutsu, Hideo
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 1096-0279
• DOI: 10.1016/j.pep.2012.02.005

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.