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1	Engineering Cell-Free Protein Expression Systems for Biotherapeutics and Biosensing Hunt, John Porter 18 March 2021 (has links) 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. Cell-free protein synthesis CFPS biotherapeutic biologic biosensor medicine endotoxin hydrofoam hormone biosensor cell-free biosensor acute lymphoblastic leukemia Engineering
2	Aminoacyl-tRNA Synthetase Production for Unnatural Amino Acid Incorporation and Preservation of Linear Expression Templates in Cell-Free Protein Synthesis Reactions Broadbent, Andrew 01 March 2016 (has links) (PDF) Proteins—polymers of amino acids—are a major class of biomolecules whose myriad functions facilitate many crucial biological processes. Accordingly, human control over these biological processes depends upon the ability to study, produce, and modify proteins. One innovative tool for accomplishing these aims is cell-free protein synthesis (CFPS). This technique, rather than using living cells to make protein, simply extracts the cells' natural protein-making machinery and then uses it to produce protein in vitro. Because living cells are no longer involved, scientists can freely adapt the protein production environment in ways not otherwise possible. However, improved versatility and yield of CFPS protein production is still the subject of considerable research. This work focuses on two ideas for furthering that research.The first idea is the adaptation of CFPS to make proteins containing unnatural amino acids. Unnatural amino acids are not found in natural biological proteins; they are synthesized artificially to possess useful properties which are then conferred upon any protein made with them. However, current methods for incorporating unnatural amino acids do not allow incorporation of more than one type of unnatural amino acid into a single protein. This work helps lay the groundwork for the incorporation of different unnatural amino acid types into proteins. It does this by using modified aminoacyl-tRNA synthetases (aaRSs), which are key components in CFPS, to be compatible with unnatural amino acids. The second idea is the preservation of DNA templates from enzyme degradation in CFPS. Among the advantages of CFPS is the option of using linear expression templates (LETs) in place of plasmids as the DNA template for protein production. Because LETs can be produced more quickly than plasmids can, using LETs greatly reduces the time required to obtain a DNA template for protein production. This renders CFPS a better candidate for high-throughput testing of proteins. However, LETs are more susceptible to enzyme-mediated degradation than plasmids are, which means that LET-based CFPS protein yields are lower than plasmid-based CFPS yields. This work explores the possibility of increasing the protein yield of LET-based CFPS by addition of sacrificial DNA, DNA which is not used as a protein-making template but which is degraded by the enzymes in place of the LETs. Andrew Broadbent cell-free protein synthesis (CFPS) unnatural amino acid incorporation aminoacyl-tRNA synthetase linear expression template (LET) aminoacylation assay sacrificial DNA Chemical Engineering
3	Designing Cell-Free Protein Synthesis Systems for Improved Biocatalysis and On-Demand, Cost-Effective Biosensors Soltani Najafabadi, Mehran 06 August 2021 (has links) 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. Mehran Soltani cell-free protein synthesis cell-free CFPS in vitro TEM-1 ?-lactamase antibiotics unnatural amino acid protein engineering site-specific covalent immobilization site-directed mutagenesis design heuristics coarse-grain simulation biosensor cell-free biosensor RNase inhibitor murine RNase inhibitor m-RI RNase inhibitor RNase A human body fluids saliva serum urine glutamine lyophilized point of care GroEL/ES folding chaperones E. coli extract Extract with RNase inhibition activity Engineering

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