Abstract

Recombinant proteins have many medical and industrial applications, but their use is complicated by commercial production and stability constraints. These issues are particularly challenging for recombinant proteins used in pharmaceutical therapeutics and clinical diagnostics. Expensive production and distribution limit the accessibility of therapeutics and diagnostics especially in the developing world. Additionally, clinical use of recombinant proteins face further challenges within biological systems including biological degradation and immunogenicity. To increase the accessibility of recombinant proteins, the cost and inefficiencies of protein manufacturing and distribution need to be significantly reduced. A powerful tool to aid in this endeavor is cell-free protein synthesis (CFPS) technology. CFPS is a versatile platform for recombinant protein production due to its open reaction environment, flexible reaction conditions, and rapid protein expression capabilities. These avoid the disadvantages of conventional manufacturing and present the capability of on-demand protein therapeutic production outside of centralized facilities. To improve the efficacy of recombinant proteins for medicinal use, protein engineering techniques such as PEGylation, or the conjugation of PEG polymers to protein surfaces, can be employed. PEGylation is widely used to enhance the pharmacokinetic properties of protein therapeutics. Deciphering optimal PEG conjugation sites is a continuing area of research that can be facilitated by CFPS systems that enable high-throughput, site-specific PEGylation. This dissertation presents advances in CFPS technology to promote increased accessibility and stability of life-saving therapeutics and diagnostics. The work presented here (1) improves on-demand therapeutic production capabilities by creating shelf-stable, endotoxin-free CFPS systems, (2) aids the rational design of next-generation PEGylated protein therapeutics through an in silico-in vitro CFPS screening platform, and (3) advances the development of portable clinical diagnostics for rapid and sustainable deployment at point-of-care through CFPS biosensor technology. The innovations of this dissertation are described in four publications. Specifically, an endotoxin-free CFPS system lyophilized with lyoprotectants is demonstrated that shows improved shelf-stability over standard lyophilized systems. A streamlined procedure for preparing endotoxin-free extract using auto-induction media is presented that significantly reduces CFPS preparation labor and time. A combinatorial screening approach is demonstrated in which coarse-grain molecular simulation informs PEGylation site selection as verified by CFPS experimental results. An inexpensive paper-based, saliva-activated CFPS biosensor platform is developed for the detection of SARS-CoV-2 sequences.

Degree

PhD

College and Department

Chemical Engineering

Rights

https://lib.byu.edu/about/copyright/

Date Submitted

2021-12-16

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd12611

Keywords

Cell-free protein synthesis, TXTL, site-specific PEGylation, protein therapeutic, biologic, unnatural amino acid, cell-free biosensor, RNA toehold switch, Covid-19, on-demand therapeutic production, point-of-care rapid diagnostic

Language

english

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Engineering Commons

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