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.
Kuruma
Yutetsu
Y
The Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
Suzuki
Toshiharu
T
Ueda
Takuya
T
D000818:Animals; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis; D008081:Liposomes; D024101:Mitochondrial Proteins / Q000096:biosynthesis; D025261:Mitochondrial Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis; D000071256:Uncoupling Protein 1
D007473:Ion Channels; D008081:Liposomes; D024101:Mitochondrial Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D000071256:Uncoupling Protein 1; C474451:F1F0-ATP synthase; C103607:mitochondrial ATPase subunit c; D025261:Mitochondrial Proton-Translocating ATPases