RECONSTITUTION OF F1-ATPASE ACTIVITY FROM ESCHERICHIA-COLI SUBUNITS ALPHA, BETA AND SUBUNIT GAMMA TAGGED WITH 6 HISTIDINE-RESIDUES AT THE C-TERMINUS

An engineered gamma subunit of Escherichia coli F-1-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 histidine s are included in the C-terminal extension, The reconstituted F-1 cont aining alpha, beta, and His-tagged gamma exhibited sixty percent of th e wild-type ATPase activity. The reconstituted alpha beta His-tag gamm a complex was subjected to affinity chromatography with nickel-nitrilo triacetic acid (Ni-NTA) agarose resin, ATPase activity mas eluted spec ifically with imidazole, These results implied that the tag sequence p rotruded to the surface of the complex and did not seriously impair th e activity. The reconstituted alpha beta His-tag gamma complex, even a fter its binding to the resin, exhibited ATPase activity suggesting th at the gamma subunit, when fixed to a solid phase, may rotate the alph a beta complex. This system may provide a nem approach for analysis of the rotation mechanisms in F-1-ATPase, (C) 1998 Federation of Europea n Biochemical Societies.