(560a) Integrating Cell-Free Protein Synthesis into Globular Protein Vesicles Towards Development of Artificial Protocells Capable of Growth and Replication

To further understand the origin of cellular life and advance biomedical technologies on a cellular level, many researchers have turned their attention towards bottom-up construction of artificial protocells. Nonetheless, one of the essential features of biological cells, growth and replication, remains challenging to mimic in many synthetic platforms as it requires complex and biologically incompatible chemistries to synthesize building block materials. Herein, we aim to leverage cell-free protein synthesis (CFPS) to develop artificial protocells capable of growth and replication in globular protein vesicle (GPV) platforms. GPVs, consisting of fully folded recombinant fusion proteins, are a promising platform for artificial protocell development due to their expected biocompatibility and spatial control over functional protein integration. GPVs are uniquely suited for engineered growth and replication via cell-free protein expression due to their building block composition, consisting exclusively of proteins. Thus, the integration of CFPS into GPV platforms would enable self-manipulation of membrane protein components via in vitro transcription and translation. To achieve this aim, we encapsulate CPFS agents including cell extracts, cofactors, genes, an energy source, and genetic material encoding recombinant proteins within the vesicle lumen. The presence of these CFPS systems within the vesicle lumen allow for de novo expression of functional proteins. Furthermore, these de novo proteins can be recombinantly engineered to integrate into the GPV membrane for programmed membrane modification post-synthesis. We demonstrate this mechanism through expression of new membrane building blocks within GPVs that integrate into the interior leaflet of the membrane, enabling growth of GPVs. This work will provide insights into artificial cell development as a major step towards modeling essential cellular functions in a synthetic platform.