Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression

Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From more than 1,200 reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 ± 0.3 g/L of protein product at the 15-μL scale (∼$55/gprotein) and 3.7 ± 0.2 g/L (∼$36/gprotein) at the 4-mL scale with oxygen supplementation. This provides an 84 to 99% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nucleotides and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of different proteins. Specifically, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will further democratize the use of cell-free systems for protein manufacturing and synthetic biology applications. ### Competing Interest Statement MLO and MCJ have filed an invention disclosure based on the work presented. MCJ has a financial interest in SwiftScale Biologics, Gauntlet Bio, Pearl Bio, Inc., Synolo Therapeutics and Stemloop Inc. MCJ's interests are reviewed and managed by Stanford University in accordance with their competing interest policies. National Science Foundation, CBET - 2341123, DGE-1842165, 2021900 DARPA, W911NF-23-2-0039 Department of Energy, DE-SC0023278