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.



College and Department

Ira A. Fulton College of Engineering and Technology; Chemical Engineering



Date Submitted


Document Type





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



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