Cell-Free Protein Synthesis of Fusion-Protein Building Blocks Enables Autonomous Growth in Globular Protein Vesicle Protocells

The capacity for autonomous synthesis of building blocks and functional molecules is a fundamental feature of living cells. Encapsulating complex synthesis systems, such as cell-free protein synthesis (CFPS), within vesicular platforms has propelled the field toward more intricate biomimicry in artificial cell development, yet synthesizing functional proteins as membrane components remains challenging. In this study, we report the successful integration of a CFPS system with globular protein vesicles (GPVs), a functionally folded protein-assembled vesicular platform. We demonstrate that in vitro transcription and translation of modular fusion proteins, which serve as GPV building blocks, facilitate the direct incorporation of newly synthesized fusion proteins into the vesicle membrane. This system supports the expression of membrane components, enabling GPVs to exhibit autonomous growth. Our approach marks substantial progress in the development of synthetic cells, providing a versatile and robust strategy to expand the available repertoire of biomimetic functions achievable through de novo protein production.