(792e) High-Yielding Escherichia Coli-Based Cell-Free Protein Synthesis of Commercially Relevant Proteins

Cell-free protein synthesis (CFPS) is an open and accessible in vitro protein production system that is highly controllable, easily probed and economical. These traits have proved useful and widely applicable for the high-throughput production, characterization and engineering of novel proteins such as oxygen-tolerant hydrogenases, signaling peptides, kinases and membrane-anchored proteins. Escherichia coli-based CFPS (eCFPS) is the most cost-efficient and is therefore commonly employed. Unfortunately, eCFPS lacks the ability to produce many commercially relevant proteins due to the absence of cofactors necessary for proper synthesis. However, commercially relevant proteins can be properly produced in eCFPS by: 1) adding proper cofactors that are exogenously produced and purified and/or 2) directly expressing proper cofactors during cell-extract preparation. Direct expression of cofactors during cell growth avoids the tedious and time-consuming steps of exogenous production and purification, and therefore lends itself to speedier and more economical high-throughput applications. However, previous reports demonstrate that yields in such systems are regrettably low. We hypothesized that yields could be increased by adjusting factors during cell extract growth and protein synthesis. By circumspectly adjusting these factors, we were able to produce two commercially relevant proteins at levels up to 7 times higher than previously reported yields. This finding further enables the economical in vitro production of complex proteins and high-throughput screening.  In addition, insights gained could help identify the optimal levels of enzyme expression for in vivo synthetic biology applications.