Dynamical Microscale Reaction Environment Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan.
Proc Natl Acad Sci U S A. 2013 Oct 15;110(42):16796-801. doi: 10.1073/pnas.1314585110. Epub 2013 Sep 30.
In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g., antibodies, enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method called liposome display that can evolve the properties of membrane proteins entirely in vitro. This method, which involves in vitro protein synthesis inside liposomes, which are cell-sized phospholipid vesicles, was applied to the pore-forming activity of α-hemolysin, a membrane protein derived from Staphylococcus aureus. The obtained α-hemolysin mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the WT. Given the ability to synthesize various membrane proteins and modify protein synthesis and functional screening conditions, this method will allow for the rapid and efficient evolution of a wide range of membrane proteins.
体外方法已经实现了蛋白质的快速高效进化，并成功产生了新型的、具有高度功能的蛋白质。然而，现有的方法仅考虑球状蛋白质（例如抗体、酶），而不考虑膜蛋白，尽管后者在生物学和药物学方面具有重要意义。在这项研究中，我们报告了一种称为脂质体展示的方法的发展，该方法可以完全在体外进化膜蛋白的特性。这种方法涉及在脂质体内部进行体外蛋白质合成，脂质体是细胞大小的磷脂囊泡，该方法应用于来自金黄色葡萄球菌的膜蛋白α-溶血素的孔形成活性。获得的α-溶血素突变体仅具有两个点突变，但与 WT 相比，其孔形成活性增加了 30 倍。鉴于能够合成各种膜蛋白以及修饰蛋白质合成和功能筛选条件，这种方法将允许广泛的膜蛋白的快速和高效进化。
