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 and enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method named liposome display that can evolve the properties of membrane proteins entirely In vitro. This method, which involves In vitro protein synthesis inside liposomes-cell-sized phospholipid vesicles-was applied to the pore-forming activity of α-hemolysin (AH), a membrane protein derived from Staphylococcus aureus. The obtained AH mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the wild type. 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.
The Exploratory Research for Advanced Technology (ERATO),
Yomo Dynamical Micro-scale Reaction Environment Project
Title: In vitro evolution of α-hemolysin using a liposome display
Authors: Satoshi Fujii, Tomoaki Matsuura, Takeshi Sunami, Yasuaki Kazuta and Tetsuya Yomo
Tetsuya Yomo Ph.D.
Professor, Graduate School of Information Science and Technology,
Department of Research Project
Japan Science and Technology Agency