Production of multi-subunit complexes on liposome through an E. coli cell-free expression system.

Recently cell-free translation systems became a laboratory research tool to obtain an objective protein. However, a general method for in vitro protein synthesis and multi-subunit complex formation on lipid membrane has not been established. Here, we describe the procedure for the production of subcomplexes of F(o)F(1)-ATP synthase using a reconstructed cell-free translation system. As for the membrane part F(o) (a(1)b(2)c((10-15))), cosynthesis of c-subunit and UncI proteins, which are integral membrane proteins, and further c (11)-ring formation in lipid bilayer are performed by use of a liposomes-containing cell-free system. Moreover, as for the cytoplasm part F(1) (alpha(3)beta(3)gammadeltaepsilon), subcomplex formations are successfully achieved by mixing the translation products.