The isolation and functional reconstitution of large membrane protein complexes is an important step towards the biochemical characterization of such sophisticated molecular machines. Reconstitution is a multistep process that requires the mild solubilization of membrane protein complexes from native membrane preparations, the purification of the complexes from protein–detergent solutions, and their incorporation into artificial phospholipid vesicles through controlled detergent removal. The major challenge is to preserve the integrity and catalytic activity of the often fragile membrane protein assemblies during the entire procedure. Here we describe the protocols for a particularly intricate example, the functional reconstitution of the mitochondrial presequence translocase (TIM23 complex). This highly versatile and dynamic protein complex is the main protein translocation machinery of the inner mitochondrial membrane and mediates the import of precursor proteins with N-terminal presequences from the cytosol.
