Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes.

The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.

Cell-free protein synthesis systems for vaccine design and production.

Vaccines are vital for protection against existing and emergent diseases. Current vaccine production strategies are limited by long production times, risky viral material, weak immunogenicity, and poor stability, ultimately restricting the safe or rapid production of vaccines for widespread utilization. Cell-free protein synthesis (CFPS) systems, which use extracted transcriptional and translational machinery from cells, are promising tools for vaccine production because they can rapidly produce proteins without the constraints of living cells, have a highly optimizable open system, and can be used for on-demand biomanufacturing. Here, we review how CFPS systems have been explored for the production of subunit, conjugate, virus-like particle (VLP), and membrane-augmented vaccines and as a tool in vaccine design. We also discuss efforts to address potential limitations with CFPS such as the presence of endotoxins, poor protein folding, reaction stability, and glycosylation to enable promising future vaccine design and production.