Reconstitution of F 1-ATPase activity from Escherichia coli subunits α, β and subunit γ tagged with six histidine residues at the C-terminus

An engineered γ subunit of Escherichia coli F 1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag γ), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F 1 containing α, β, and His-tagged...

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Bibliographic Details
Published in:FEBS letters 1998-05, Vol.427 (1), p.64-68
Main Authors: Ekuni, Atsuko, Watanabe, Hikaru, Kuroda, Nozomi, Sawada, Ken, Murakami, Hiroshi, Kanazawa, Hiroshi
Format: Article
Language:English
Subjects:
F 1-ATPase
Fusion subunit
Reconstitution
Rotation mechanism
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Summary:An engineered γ subunit of Escherichia coli F 1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag γ), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F 1 containing α, β, and His-tagged γ exhibited sixty percent of the wild-type ATPase activity. The reconstituted αβHis-tag γ complex was subjected to affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin. ATPase activity was eluted specifically with imidazole. These results implied that the tag sequence protruded to the surface of the complex and did not seriously impair the activity. The reconstituted αβHis-tag γ complex, even after its binding to the resin, exhibited ATPase activity suggesting that the γ subunit, when fixed to a solid phase, may rotate the αβ complex. This system may provide a new approach for analysis of the rotation mechanisms in F 1-ATPase.
ISSN:0014-5793
1873-3468
DOI:10.1016/S0014-5793(98)00395-0