In vitro directed evolution of alpha-hemolysin by liposome display

We have developed a method to enable in vitro directed evolution that can be applied to membrane proteins. This method, termed liposome display, uses liposomes as compartments in which membrane proteins are synthesized and as scaffolds for membrane protein integration. Thus, the synthesized membrane proteins are displayed on the surface of the liposome and exhibit their functions. A randomly mutated DNA library of the membrane protein was generated, encapsulated in the liposomes at the single-molecule level, and used to generate a liposome library. Liposomes displaying the desired membrane protein function were selected, thus accumulating the DNA molecule encoding the desired membrane protein. We have applied this method to alpha-hemolysin, a membrane protein derived from Staphylococcus aureus. Alpha-hemolysin forms a nanopore in the membrane, which allows the penetration of small molecules. We aimed to improve this nanopore activity by using the liposome display method. Consequently, alpha-hemolysin evolved and attained a higher specific affinity for the liposome membrane. In this review, we describe the essential characteristics of liposome display and the properties of the evolved alpha-hemolysin obtained by this method.