Kuruma Yutetsu, Suzuki Toshiharu, Ueda Takuya
The Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
Methods Mol Biol. 2010;607:161-71. doi: 10.1007/978-1-60327-331-2_14.
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.
最近，无细胞翻译系统成为获取目标蛋白质的实验室研究工具。然而，尚未建立在脂质膜上进行体外蛋白质合成和多亚基复合物形成的通用方法。在此，我们描述了使用重建的无细胞翻译系统生产F(o)F(1)-ATP合酶亚复合物的程序。对于膜部分F(o)（a(1)b(2)c((10 - 15))），通过使用含脂质体的无细胞系统进行整合膜蛋白c亚基和UncI蛋白的共合成，以及在脂质双层中进一步形成c(11)环。此外，对于细胞质部分F(1)（alpha(3)beta(3)gammadeltaepsilon），通过混合翻译产物成功实现了亚复合物的形成。
