Purification and Reconstitution of Ilyobacter tartaricus ATP Synthase

F-type Adenosine triphosphate (ATP) synthase is a membrane-bound macromolecular complex, which is responsible for the synthesis of ATP, the universal energy source in living cells. This enzyme uses the proton- or sodium-motive force to power ATP synthesis by a unique rotary mechanism and can also operate in reverse, ATP hydrolysis, to generate ion gradients across membranes. The F₁F_o-ATP synthases from bacteria consist of eight different structural subunits, forming a complex of ~550 kDa in size. In the bacterium Ilyobacter tartaricus, the ATP synthase has the stoichiometry α₃β₃γδεab₂c₁₁. This chapter describes a wet-lab working protocol for the purification of several tens of milligrams of pure, heterologously (E. coli-) produced I. tartaricus Na⁺-driven F₁F_o-ATP synthase and its subsequent efficient reconstitution into proteoliposomes. The methods are useful for a broad range of subsequent biochemical and biotechnological applications.