F1‐ATPase (F1) is a multisubunit water‐soluble domain of FoF1‐ATP synthase and is a rotary enzyme by itself. Earlier genetic studies using yeast suggested that two factors, Atp11p and Atp12p, contribute to F1 assembly. Here, we show that their mammalian counterparts, AF1 and AF2, are essential and sufficient for efficient production of recombinant bovine mitochondrial F1 in Escherichia coli cells. Intactness of the function and conformation of the E. coli‐expressed bovine F1 was verified by rotation analysis and crystallization. This expression system opens a way for the previously unattempted mutation study of mammalian mitochondrial F1.
