(402c) Integration of Cell-Free Protein Expression with Recombinant Fusion Protein Assemblies

To better understand the complex mechanisms that enable life on a microscopic level, biological processes can be simplified in engineered materials via the bottom-up construction of biomimetic artificial cells. Globular protein vesicles (GPVs), consisting of fully folded recombinant fusion proteins, are promising for artificial cell development due to their highly specific control over protein type, orientation, and proportion in the vesicle membrane. An advanced GPV-based artificial cell model can be achieved through the integration of cell-free protein expression mechanisms, as previously demonstrated in synthetic liposomes as a major step towards modeling the essential functions carried out by proteins in living cells. Furthermore, the integration of cell-free protein synthesis (CFPS) into GPV platforms would enable self-manipulation of membrane protein components via in vitro transcription and translation. Preliminary data has indicated that the presence of CFPS agents, including cell extracts, cofactors, genes, and an energy source, disrupts self-assembly of amphiphilic recombinant protein building blocks into GPVs. We aim to realize cell-free protein expression in GPVs through developing engineering strategies that can non-destructively interface CFPS systems with fusion protein assemblies. I will discuss the sequestration of CFPS systems in hydrogel particles that allow protein expression within fusion protein assemblies along with tuning hydrogel properties for controlled degradation, release, and fouling properties. This work will provide insights into the filed of artificial cell development by introducing autonomous production of vesicle membrane constituents for modifications and additional functions in cell-free synthetic systems.