CAREER: Engineering Self-Assembly of Recombinant Fusion Proteins for Bottom-Up Construction of Protein-Powered Synthetic Protocells

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This Faculty Early Career Development Program (CAREER) project aims to develop a synthetic protocell constructed of protein building blocks. Synthetic protocells, or primitive versions of cells, can serve as simple, precursor models to living cells and can advance understanding about the basic rules of life. The research objective for this project is to develop design strategies to control the properties of new classes of protein-powered synthetic protocells. The proposed protocells will be created by self-assembly of engineered proteins, resulting in cell-like structures exhibiting specific activities. Ultimately, the synthetic protocells will serve as smart, autonomous cell-like particles that can be used for a wide range of applications from protein delivery systems to micro-bioreactors. This research project will be coupled with educational efforts to provide hands-on learning opportunities to underrepresented students at different levels and to develop academic curricula on the self-assembly of recombinant fusion proteins.The research objective of this CAREER project is to create a new class of synthetic cells composed of functionally folded, globular fusion proteins with controlled structural, mechanical, and biological properties. The globule-leucine zipper-elastin like polypeptide (GLE) fusion protein complexes to be studied serve as basic building blocks to make globular protein vesicles (GPVs). The biophysical properties of protein building blocks will be engineered to control the self-assembly and function of GPVs. This project combines vesicle assembly of diverse GLE proteins, with precise control over vesicle mechanical and structural properties, and de novo protein synthesis inside GPVs to mimic essential cellular functions including self-growth and cascade reactions. The specific objectives are to 1) elucidate the physicochemical role of globular proteins in vesicle self-assembly, 2) understand the structure-property-performance relationships of GPVs to engineer basic cellular membrane functions such as deformability and fusion dynamics, and 3) functionalize GPVs through cell-free protein synthesis and enzymatic cascade reactions towards an autonomous synthetic cell. Recombinant protein technology, thermodynamics of block copolymer assembly in dilute solutions, and soft materials engineering and characterization tools will be applied to achieve these objectives.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.