Development of Expression System for Photoreceptor Using Cell-free Protein Synthesis and Application of Its system for Their Functional Analysis

一般来说，高产量的膜蛋白的功能性无细胞蛋白质合成是困难的。在本研究中，我们开发了一种新的无细胞蛋白质合成技术，即通过透析法同时进行类固醇类洗涤剂的去除、脂双层的形成和蛋白质的合成。我们在大量(0.5 mg/mL反应液)中获得了功能性细菌视紫红质，其产率是以前报道的制剂的5-25倍。此外，它还成功地实现了利用活细胞表达系统无法大量获得的海藻和真细菌视紫红质的功能性过表达。

In general, a functional cell-free protein synthesis for membrane proteins at high yield is difficult. In the present study, we developed the novel cell-free protein synthesis technology that was performed the steroid derived detergent removal, the lipid bilayer formation, and the protein synthesis by the dialysis method at the same time. We obtained the functional Bacteriorhodopsin in a large amount (0.5mg/mL reaction solution), and this yield was 5-25 times higher than previously described preparations. In addition, it succeeded a functional overexpression of the rhodopsin from marine algae and eubacteria that could not be obtained in large amount using the living cell expression system.