Giant unilamellar vesicles (GUVs) are powerful biomimetic models for studying membrane proteins, yet their use is hampered by challenges in protein reconstitution, including vesicle fragility and inefficient insertion. Traditional detergent- or fusion-based insertion methods using purified membrane proteins can compromise the system by destabilizing vesicles and altering lipid compositions. While in situ cell-free protein synthesis (CFPS) offers a promising alternative, the efficiency of spontaneous protein insertion is critically dependent on the surrounding lipid composition. This creates a major bottleneck, as conventional GUV technologies lack the throughput needed to screen the vast combinatorial space of lipid environments to find optimal conditions for protein insertion and function. To address this gap, our microfluidic platform is designed for parallel analysis of protein insertion across a library of GUVs with diverse, physiologically relevant lipid compositions. We first fabricate mechanically robust GUVs using octanol-assisted liposome assembly (OLA) with an internal hydrogel scaffold, which allows us to precisely control membrane properties like charge, order, and asymmetry, while an internal hydrogel scaffold enhances stability. These GUVs are then immobilized in microwell trapping devices to minimize shear stress, creating stable arrays. With the GUV library in place, CFPS reagents are applied externally, enabling high-throughput screening of insertion conditions as proteins are synthesized. This approach allows for systematic screening of how specific lipid environments affect membrane protein insertion. Our platform provides a powerful and scalable tool for functional screening and advancing the fundamental understanding of protein-lipid interactions.
Noh et al. (Sun,) studied this question.