Jeremy Hunt, Brigham Young University
Engineering
Unnatural amino acid incorporation is a power tool in the synthetic biology toolbox that allows for unique residue chemistry to be incorporated into proteins. This technology has many promising applications in areas such as protein-protein interaction, biotherapeutics, biosensing, and biocatalysis. One challenge of uAA-incorporation is the current inability to properly predict the impact of the novel uAA-residue chemistry on proper protein folding and function. Therefore, a screening technology would be desirable to rapidly assess the viability of uAA-incorporation sites. Cell-free protein synthesis (CFPS) provides a promising basis for rapid screening technologies. The open environment of CFPS has a variety of advantages over conventional in vivo systems, such as direct monitoring, selective protein expression, and facile deployment of synthetic pathways. Another primary benefit CFPS has over in vivo expression is the ability to directly express proteins from PCR-generated products, known as linear expression templates (LETs). The use of LETs decreases the labor and time to expression of recombinant proteins. Thus, LET-based CFPS is a propitious system for rapid screening of uAA-incorporation. Here we demonstrate uAA-incorporation using LET-based CFPS and identify how this technology can significantly reduce time and labor to rapidly express proteins containing uAAs.