| Cell-free protein synthesis (CFPS) is a rapidly developing field with a wide range of applications spanning bio-manufacturing to molecular biology. Although there have been considerable advances in CFPS technology over the last few decades, there remains a need to develop a practical and robust eukaryotic CFPS platform that can synthesize a large variety of active complex eukaryotic proteins. In this document, I outline several strategies toward accomplishing this goal through the development of a eukaryotic CFPS platform based on Saccharomyces cerevisiae extract. This project encompasses a wide range of technical disciplines and skill sets, including bioprocess engineering and molecular biology. Our initial work involved developing standardized extract preparation procedures, followed by CFPS reaction optimization and characterization. With a functional platform in place, we were able to co-activate natural energy metabolism to power the reaction and to further use this system to enhance our understanding of translation initiation mechanisms. Lastly, we demonstrate that this platform can be used to synthesize pharmaceutically relevant products with high solubility and opened the door for a number of valuable future applications. |
