Membrane Formulation Impacts Vesicle-Based Artificial Cell Functions

Engineering vesicle-based artificial cells capable of mimicking cellular processes is a central objective in synthetic biology. However, while giant unilamellar vesicles (GUVs) have become a popular artificial cell platform, the impact of fundamental design features remains obscure. Here, we assembled GUVs composed primarily of two phospholipids: POPC, a phosphatidylcholine widely employed in GUV preparation, and DOPC, a phosphatidylcholine with a lower melting temperature and higher fluidity. We found that the phospholipid mixture leads to an increase in average GUV size (compared to POPC only) without compromising GUV yield (in contrast to DOPC only). Moreover, we studied two artificial cell functions: enzyme-driven protein-membrane docking and protein expression. Artificial cells prepared from the phospholipid mixture exhibited enhanced relocation of proteins to the membrane and enhanced protein expression. Furthermore, our data allowed us to identify the limiting factor that underlies the observed differences in protein expression between membrane formulations: the efficiency of macromolecular encapsulation. Altogether, our study underscores the importance of membrane engineering as a powerful strategy for advancing the rational development of artificial cells with functional capabilities.