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

该学院早期职业发展计划(Career)项目旨在开发一种由蛋白质构建块构成的合成原始细胞。合成的原细胞，或细胞的原始版本，可以作为活细胞的简单前体模型，并可以促进对生命基本规则的理解。这个项目的研究目标是开发设计策略来控制新型蛋白质动力合成原始细胞的性质。被提议的原细胞将通过工程蛋白的自我组装而产生，从而产生具有特定活动的类细胞结构。最终，合成的原始细胞将成为智能的、自主的细胞状颗粒，可用于从蛋白质输送系统到微型生物反应器的广泛应用。这项研究项目将与教育努力相结合，为不同水平的未被充分代表的学生提供实践学习机会，并开发重组融合蛋白自组装的学术课程。这一职业项目的研究目标是创造一种新的合成细胞，由具有可控结构、机械和生物特性的功能折叠的球形融合蛋白组成。所研究的球状-亮氨酸拉链-类弹性蛋白多肽(GLE)融合蛋白复合体是构成球状蛋白囊泡(GPV)的基本构件。将对蛋白质构建块的生物物理性质进行工程设计，以控制GPV的自组装和功能。该项目结合了不同GLE蛋白的囊泡组装，以及对囊泡机械和结构特性的精确控制，以及在GPV内的从头合成蛋白质，以模拟基本的细胞功能，包括自我生长和级联反应。其具体目标是1)阐明球状蛋白在囊泡自组装中的物理化学作用，2)了解GPV的结构-性质-性能关系，以设计基本的细胞膜功能，如变形性和融合动力学，以及3)通过无细胞蛋白质合成和酶促合成细胞的级联反应使GPV功能化。重组蛋白质技术、嵌段共聚聚合物在稀溶液中组装的热力学以及软材料工程和表征工具将被用于实现这些目标。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.