서강대학교

초록

Cell-free protein synthesis (CFPS) based on cell lysate has many advantages over protein synthesis in vivo. In particular, the lack of cell walls allows direct manipulation of biochemical cascades that usually occur in the cytoplasmic space. Meanwhile, we recapitulate that when cell lysate is encapsulated in artificial cell membranes, it could be the ideal cytoplasm for artificial cells capable of protein synthesis and tremendous amounts of biological reactions. It was, however, difficult to implement CFPS reactions using the cell lysates due to the osmotic instability in the confined spaces provided by liposomes and inhomogeneity of the cell lysate. In this research, we developed efficient and sustainable cell lysate-based CFPS system in a giant unilamellar vesicle (GUV). We first selected appropriate sugars for inner and outer buffer to keep the osmotic pressure as low as possible during the whole reaction. For GUV formation, emulsion transfer method was adopted to prepare the cell-lysate encapsulated GUV with high encapsulation efficiency. By applying ultrasound to the vesicles, the homogeneity of the encapsulated cell lysis solution was extremely increased, and as a result, almost all GUVs showed sharp green fluorescence. To keep GUV intact against unpredictable change of osmotic pressure with enhanced stability, saturated lipids and high amounts of cholesterol were added with lipid phase separation. Finally, we successfully fabricated artificial cells as a bioreactor, which are feasible to synthesize various proteins inside using a lysate-based expression system and possible to last for 14 hours at which the reaction in vitro reaches the plateau.