Engineering Cell-Free Biosystems for On-Site Production and Rapid Design of Next-Generation Therapeutics

• Main Author: Wilding, Kristen Michelle
• Format: Others
• Published: BYU ScholarsArchive 2018
• Subjects: synthetic biology; cell-free protein synthesis; endotoxin removal; lyophilization; lyoprotectant; unnatural amino acid; high-throughput screening; Engineering
• Online Access: https://scholarsarchive.byu.edu/etd/7713; https://scholarsarchive.byu.edu/cgi/viewcontent.cgi?article=8713&context=etd

While protein therapeutics are indispensable in the treatment of a variety of diseases, including cancer, rheumatoid arthritis, and diabetes, key limitations including short half-lives, high immunogenicity, protein instability, and centralized production complicate long-term use and on-demand production. Site-specific polymer conjugation provides a method for mitigating these challenges while minimizing negative impacts on protein activity. However, the location-dependent effects of polymer conjugation are not well understood. Cell-free protein synthesis provides direct access to the synthesis environment and rapid synthesis times, enabling rapid evaluation of multiple conjugation sites on a target protein. Here, work is presented towards developing cell-free protein synthesis as a platform for both design and on-demand production of next generation polymer-protein therapeutics, including (1) eliminating endotoxin contamination in cell-free reagents for simplified therapeutic preparation, (2) improving shelf-stability of cell-free reagents via lyophilization for on-demand production, (3) coupling coarse-grain simulation with high-throughput cell-free protein synthesis to enable rapid identification of optimal polymer conjugation sites, and (4) optimizing cell-free protein synthesis for production of therapeutic proteins