Degree

Doctor of Philosophy (PhD)

Department

The Department of Biological and Agricultural Engineering

Document Type

Dissertation

Abstract

Proteins are highly structural polymers, performing an enormous range of roles within organisms. Unlike synthetic polymers, proteins have a significant hierarchical ordering that can be disrupted by many environmental factors, which limits the scale-up manufacturing capability as well as the utilization of proteins as therapeutic agents. In this study, the Cell-Free Protein Synthesis (CFPS) system was developed to enhance the production capacity and applications of the therapeutic proteins. The CFPS is an advanced biosynthetic and genetic-engineered system. It enables the efficient production of proteins when the conventional cell-based protein synthesis systems cannot be applied, and when sophisticated control is required for protein synthesis and folding. The nanosized hydrogels were designed as a protein delivery vehicle and its protein loading ability was tested.

The main goal of this study is to develop a platform for the biomanufacturing and transport of proteins for therapeutic purposes. In this study the customizable CFPS system was demonstrated as a next-generation platform for therapeutic protein production. The human skin protein, monomeric filaggrin was selected as a model protein to be translated in the developed CFPS platform. This study demonstrated that a tailored cell-free system is an effective platform for human protein synthesis with high production yield. The therapeutic protein filaggrin expression was significantly improved in a tailored platform compared to that of non-optimized CFPS condition, representing 28 ± 5 µM of soluble protein yield. In this study we present the synthesis of gelatin methacryloyl, which can be fabricated from gelatin with a wide range of methacrylic substitution. The study characterized the physical properties of nanogels, swelling ability, size distribution, and the protein binding and release. This study demonstrated that modulation of nanogels matrix crosslinking density via photopolymerization enables great control about swelling ability of nanogels matrix and protein release.

These studies demonstrated the opportunity of utilizing the CFPS system to produce functional proteins. It was tailored for the engineered form of therapeutic proteins with high specificity. Nanogels delivery system presented here provided a robust platform for controlled therapeutic protein binding and release.

Committee Chair

Kwon, Yongchan

Available for download on Monday, January 15, 2024

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