Engineering Cell-Free Protein Synthesis Within Protocells

Cell-free protein synthesis (CFPS) has emerged as a versatile platform for producing proteins in vitro with exceptional programmability, rapid prototyping capability, and an open biochemical environment. However, conventional CFPS is typically performed in homogeneous solutions that lack the spatial organization, molecular confinement, and dynamic regulation characteristic of living cells. Integrating CFPS with protocells overcomes these limitations by recreating cell-like microenvironments that enable compartmentalized gene expression and increasingly life-like biological functions. In this review, we summarize recent advances in protocell platforms for CFPS and discuss how compartmentalization influences the efficiency, regulation, and functionality of protein synthesis. We compare the major protocell platforms and highlight the physicochemical principles governing confined protein synthesis, such as molecular crowding, selective permeability, molecular partitioning, and phase separation. We then review strategies for enhancing protein production and constructing life-like behaviors, including programmable gene expression, membrane protein synthesis, DNA replication, membrane growth, morphogenesis, division, and intercellular communication. Finally, we discuss emerging applications in biosensing, protein engineering, and therapeutics, and outline future challenges and opportunities for developing protein-producing protocells toward living systems.