A Detailed Protocol for Preparing Millimeter-sized Supergiant Liposomes that Permit Efficient Eukaryotic Cell-free...

Liposomes have been used as a pseudo cell membrane for encapsulating biomolecules and creating an artificial cell in the interior where biochemical reactions can occur. Among the several methods used to prepare biomolecule-encapsulating liposomes, the spontaneous emulsion transfer method is superior to others in that it allows us to readily prepare relatively large liposomes whose sizes are controlled (from micrometer- to millimeter-sized liposomes) without special equipment. However, conventional protocols for this method require liposomes to contain a considerably high concentration of sucrose (high-density solute), which severely inhibits gene expression, one of the most important biochemical reactions. Thus, we optimized the preparation conditions to develop a wheat germ extract (WGE)-based protocol that requires a much lower concentration of sucrose and has almost no effect on eukaryotic cell-free translation. Our protocol allows us to successfully prepare millimeter-sized, moderately stable, WGE-encapsulating liposomes in which WGE translation takes place efficiently. Since a broad range of genes derived from various types of organisms can be efficiently translated in a WGE-based translation system, liposomes prepared using our protocol would be useful as a versatile research tool for artificial cells.