Overproduction of a functional A1 ATPase from the archaeon Methanosarcina mazei Go1 in Escherichia coli

Single subunits of the A(1) ATPase from the archaeon Methanosarcina mazei Gol were produced in E. coli as MalE fusions and purified, and polyclonal antibodies were raised against the fusion proteins. A DNA fragment containing the genes ahaE, ahaC, ahaF, ahaA, ahaB, ahaD, and ahaG, encoding the hydrophilic A(1) domain and part of the stalk of the A(1)A(O) ATPase of M. mazei Gol, was constructed, cloned into an expression vector and transformed into different strains of Escherchia coli. In any case, a functional, ATP-hydrolysing A(1) ATPase was produced. Western blots demonstrated the production of subunits A, B, C, and F in E. coli, and minicell analyses suggested that subunits D, E, and G were produced as well. This is the first demonstration of a heterologous production of a functional ATPase from an archaeon. The A(1) ATPase was sensitive to freezing but lost only about 50% of its activity within 18 days on ice. Inhibitor studies revealed that the heterologously produced A(1) ATPase is insensitive to azide, dicyclohexylcarbodiimide and bafilomycin A(1), but sensitive to diethylstilbestrol and its analogues dienestrol and hexestrol. The expression system described here will open new avenues towards the functional and structural analyses of this unique class of enzymes.