Freeze‐thaw Fusion Enables Multi‐component Delivery and Protein Synthesis in POPG‐containing Lipid Vesicles

Fusion between giant unilamellar vesicles (GUVs) provides a simple way to deliver and mix biochemical substrates within compartments surrounded by phospholipid membrane. Negatively charged phospholipids such as 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) (POPG) have recently been shown to improve vesicle robustness during freeze‐thaw (F/T) treatments by suppressing membrane rupture and reducing leakage of encapsulated contents. Here we show that incorporating POPG into phospholipid membranes markedly enhances macromolecular encapsulation and supports efficient cell‐free protein synthesis using the reconstituted PURE translation system within GUVs. Moreover, we demonstrate that a single F/T‐mediated fusion event is sufficient to deliver the dozens of protein components required for the PURE system into preformed vesicles, enabling robust protein synthesis after fusion. POPG‐containing membranes yielded higher protein output and a larger fraction of translation‐active compartments, consistent with improved substrate retention during the freezing process. These results establish freeze‐thaw fusion as a useful method for supplying complex biochemical components into phospholipid vesicles. This strategy expands the functional capabilities of artificial cell models and provides a general framework for constructing artificial compartments capable of sustained gene expression and multi‐component biochemical activity.