title: Reconstitution of F1-ATPase activity from Escherichia coli subunits alpha, beta and subunit gamma tagged with six histidine residues at the C-terminus.
abstract: An engineered gamma subunit of Escherichia coli F1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag gamma), respectively, was overproduced in E. coli and purified. Six histidines are included in the C-terminal extension. The reconstituted F1 containing alpha, beta, and His-tagged gamma exhibited sixty percent of the wild-type ATPase activity. The reconstituted alphabeta His-tag gamma 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 alphabeta His-tag gamma complex, even after its binding to the resin, exhibited ATPase activity suggesting that the gamma subunit, when fixed to a solid phase, may rotate the alphabeta complex. This system may provide a new approach for analysis of the rotation mechanisms in F1-ATPase.
authors.affiliation: Department of Biotechnology, Faculty of Engineering, Okayama University, Tsushimanaka, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Ekuni
authors.forename: H
authors.initials: H
authors.lastname: Watanabe
authors.forename: N
authors.initials: N
authors.lastname: Kuroda
authors.forename: K
authors.initials: K
authors.lastname: Sawada
authors.forename: H
authors.initials: H
authors.lastname: Murakami
authors.forename: H
authors.initials: H
authors.lastname: Kanazawa
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D015202:Protein Engineering; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006639:Histidine; D006180:Proton-Translocating ATPases