Unnatural amino acids (uAA) expand the structural and functional possibilities of proteins. Numerous previous studies have demonstrated uAA as a powerful tool for protein engineering, but challenges also remain. Three notable such challenges include: (1) the fitness of uAA-incorporated proteins are difficult to predict and time-consuming to screen with conventional methods, (2) uAA incorporation in difficult-to-express proteins (e.g. membrane proteins such as G-protein coupled receptors) remain challenging, and (3) the incorporation of multiple types of uAA are still limited. In response, we pose cell-free protein synthesis (CFPS), a rapid and versatile in vitro expression system, as a platform to explore solutions to these challenges. The "cell-free" nature of CFPS enables it to accelerate protein expression and tolerate extensive modifications to its translational environment. In this work, these advantages were utilized to address the aforementioned challenges by: (1) rapidly expressing and screening uAA-containing proteins, (2) incorporating uAA in functional G-protein coupled receptor in the presence of membrane-mimicking lipid additives, and (3) engineer the translational environment extensively towards multiple uAA incorporation.
College and Department
Ira A. Fulton College of Engineering and Technology; Chemical Engineering
BYU ScholarsArchive Citation
Schinn, Song Min, "Cell-Free Synthesis of Proteins with Unnatural Amino Acids: Exploring Fitness Landscapes, Engineering Membrane Proteins and Expanding the Genetic Code" (2017). Theses and Dissertations. 6496.
Synthetic Biology, cell-free protein synthesis, unnatural amino acid, non-natural amino acid, high throughput screening, linear DNA expression template, membrane proteins, G-protein coupled receptors, codon reassignment