Cell-free protein synthesis is an emerging technology that has many applications. The open nature of this system makes it a compelling technology that can be manipulated to answer many needs that are unavailable in other systems. This dissertation reports on engineering this technology for: 1) sense codon emancipation for incorporation of multiple unnatural amino acids; 2) expressing a hard-to-express anticancer biotherapeutic and introducing a just-add-water system; 3) a biosensing ligand that interacts with nuclear hormone receptors. Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A promising approach to sense codon emancipation is the targeted in vitro removal of native tRNA. Here we introduce a new in-vitro or "cell-free" approach to emancipate sense codons via efficient and affordable degradation of endogenous tRNA using RNase-coated superparamagnetic beads. The presented method removes greater than 99% of tRNA in cell lysates, while preserving cell-free protein synthesis activity. The resulting tRNA-depleted lysate is compatible with in vitro-transcribed synthetic tRNA for the production of peptides and proteins. Biotherapeutics have many promising applications, such as anti-cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. In this work, we demonstrate the use of cell-free systems for the expression and direct screening of the difficult-to-express cytotoxic protein onconase. Using cell-free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a "just-add-water" lyophilized cell-fee system to produce onconase. Here we introduce a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The biosensor is based on an engineered, allosterically-activated fusion protein, which contains the ligand binding domain from a target NHR. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.
College and Department
Ira A. Fulton College of Engineering and Technology; Chemical Engineering
BYU ScholarsArchive Citation
Salehi, Sayed Mohammad, "Engineering Cell-free Protein Synthesis Technology for Codon Reassignment, Biotherapeutics Production using Just-add-Water System, and Biosensing Endocrine Disrupting Compounds" (2017). All Theses and Dissertations. 6711.
Sayed Mohammad Amin Salehi, cell-free protein synthesis, codon emancipation, cancer biotherapeutics, endocrine disrupting compounds, nuclear hormone receptors, biosensor