Abstract

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.

Degree

PhD

College and Department

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

Rights

http://lib.byu.edu/about/copyright/

Date Submitted

2017-08-01

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd9451

Keywords

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

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