Therapeutic proteins have become a cornerstone of modern medicine since the FDA approval of recombinant human insulin in 1982. Likewise, biosensors transform chemical detection and disease diagnostics by identifying biomarkers, chemical contaminants, and infective agents. Long-standing methods for creating therapeutics and biosensors employ whole cells such as Escherichia coli (E. coli). Alternatively, cell-free protein synthesis (CFPS) employs the enzymatic reactions necessary for protein production and biosensing within a cell, but in an engineered reactor environment facilitating unprecedented access to and control over biochemical machinery, preservation by cryodesiccation for portable deployment, and functionality in cytotoxic applications. This dissertation reports advances in an E. coli CFPS production platform toward creating therapeutic proteins by this means. First, an endotoxin-free CFPS platform is created by optimizing fermentation and cell-extract harvest of an endotoxin-free E. coli strain. Next, liquid cell growth culture media is specially formulated to change chemical composition during cell culture and provide a streamlined method for producing high-yielding, endotoxin-free E. coli CFPS. Then, novel CFPS bioreactor formats are mathematically validated and developed which employ "hydrofoam" and oxygen to increase therapeutic protein production yield. Additionally, advances are reported in CFPS biosensing technology. First, a chimeric fusion protein incorporating the ligand binding domain of the human estrogen receptor is expressed in CFPS to detect estrogenic chemicals in the presence of human blood and urine. Next, the molecular mechanism of this protein construct is elucidated and the assay readout is optimized with mathematical simulations and CFPS. Then, CFPS is metabolically engineered to create a biosensor of L-glutamine, the most abundant amino acid in the body. Finally, this dissertation reports the development of a synergistic platform for potentially treating Acute Lymphoblastic Leukemia wherein CFPS is engineered to both produce the therapeutic protein crisantaspase and assess its activity in the presence of human serum for improved, potentially even personalized treatment of the disease. It is anticipated that the advances reported herein will contribute to the utility of in vitro or cell-free protein synthesis for therapeutic and diagnostic applications.

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.

The open nature of Cell-Free Protein Synthesis (CFPS) systems has enabled flexible design, easy manipulation, and novel applications of protein engineering in therapeutic production, biocatalysis, and biosensors. This dissertation reports on three advances in the application of CFPS systems for 1) improving biocatalysis performance in industrial applications by site-specific covalent enzyme immobilization, 2) expressing and optimizing a difficult to express a mammalian protein in bacterial-based CFPS systems and its application for cost-effective, on-demand biosensors compatible with human body fluids, and 3) streamlining the procedure of an E. coli extract with built-in compatibility with human body fluid biosensors. Site-specific covalent immobilization stabilizes enzymes and facilitates recovery and reuse of enzymes which improves the net profit margin of industrial enzymes. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. This dissertation reports the reliability of several design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of a target enzyme, TEM-1 ?-lactamase. This work demonstrates that the design heuristics can successfully identify a subset of favorable locations for experimental validation. This approach highlights the advantages of combining coarse-grain simulation and high-throughput experimentation using CFPS to efficiently identify optimal enzyme immobilization sites. Additionally, this dissertation reports high-yield soluble expression of a difficult-to-express protein (murine RNase Inhibitor or m-RI) in E. coli-lysate-based CFPS. Several factors including reaction temperature, reaction time, redox potential, and presence of folding chaperones in CFPS reactions were altered to find suitable conditions for m-RI expression. m-RI with the highest activity and stability was used to develop a lyophilized CFPS biosensor in human body fluids which reduced the cost of biosensor test by ~90%. Moreover, an E. coli extract with RNase inhibition activity was developed and tested which further streamlines the production of CFPS biosensors compatible with human body fluids.