Owen Koucky
Faculty Sponsor: Malcolm Campbell
Cell-free protein synthesis (CFPS) has the potential to improve pharmaceutical production by reducing physiological and evolutionary constraints of in vivo systems. However, concerns about the scalability of CFPS prevent current large-scale producers from adopting in vitro systems, so in vivo expression remains the gold standard for industrial protein synthesis. We are optimizing CFPS to be scalable for industrial applications. In optimizing CFPS from literature protocols, we offer an improved method for making the amino acid solution as well as the incorporation of a 15- to 30-minute pre-DNA incubation step which enhances CFPS output. Additionally, we present compelling evidence for conducting buffer optimization experiments for each batch of E. coli lysate used in CFPS. We demonstrated enhanced CFPS output when we reduced the concentration of the polyethylene glycol-6000 crowding factor by half. Finally, we report a decline in pH throughout the CFPS incubation period that we hypothesize is necessary for the initiation of CFPS. We have incorporated all of our modifications in a comprehensive protocol for reliable and robust CFPS.
