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 and not membrane proteins. 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 a-hemolysin (AH), a membrane protein derived from Staphylococcus aureus. Schematic of the liposome display is shown in figure below. (A)Gene encoding WT AH was subjected to mutagenesis to generate a randomly mutagenized gene library. (B) Gene was encapsulated in liposomes at the single-molecule level, together with the cell free translation (PURE) system. (C) After liposome preparation, AH was synthesized and the fluorescence ligand (AF488) was added to the exterior of the liposomes. The ligand was cell-impermeable due to its negative charge and was expected to penetrate the membrane only through the AH pores. (D) Liposomes with high-fluorescence signals were collected via fluorescent activated cell sorter (FACS). (E) Finally, the recovered gene was amplified and included in the next round of selection. Eventually, the obtained AH mutant possessed two point mutations and exhibited a 30-fold increase in its pore-forming activity compared with the WT.[Fujii et al., PNAS 2013 110 (42) 16796]

In vitro evolution of membrane protein, a-hemolysin using a liposome display

247th National Meeting (2014)

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