Expression of the wild-type and the Cys-/Trp-less alpha 3 beta 3 gamma complex of thermophilic F1-ATPase in Escherichia coli.
The alpha, beta and gamma subunits of F1-ATPase from thermophilic Bacillus PS3 were expressed in Escherichia coli cells simultaneously in large amounts. Most of the expressed subunits assembled into a form of alpha 3 beta 3 gamma complex in E. coli cells and this complex was easily purified to homogeneity. The recombinant alpha 3 beta 3 gamma complex thus obtained showed similar enzymatic properties to the alpha 3 beta 3 gamma complex obtained by in vitro reconstitution from individual subunits (Yokoyama, K. et al. (1989) J. Biol. Chem. 264, 21837-21841) except that the former had several-fold higher ATPase activity than the latter. Using this expression system, a mutant alpha 3 beta 3 gamma complex with no Trp and Cys was generated by replacing alpha Cys193 and alpha Trp463 with Ser and Phe, respectively. This mutant complex was functionally intact, indicating both residues are not essential for catalysis. The Cys-/Trp-less complex is a convenient 'second wild type' enzyme from which one can generate mutants with Trp (as a fluorescent probe) or Cys (as an acceptor of a variety of probes) at desired positions without concern for 'background' Trp and Cys residues.
Matsui
T
T
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Yokohama, Japan.
Yoshida
M
M
D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases