Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 82, Issue 2, Pages 396-401Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.02.005
Keywords
ATP synthase; c-Subunit ring; Membrane protein purification; New His-tag; Proton-translocation activity; Solid-state NMR
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Funding
- Targeted Proteins Research Program
- Korean Research Foundation
- Korea government, MEST
- Grants-in-Aid for Scientific Research [22770108] Funding Source: KAKEN
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FoF1-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F portion is responsible for the H+ translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the gamma subunit of F-1. For solid-state NMR measurements, FoF1 of thermophilic Bacillus PS3 (TFoF1) 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. (C) 2012 Elsevier Inc. All rights reserved.
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