Abstract 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 1, 231 different 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 (~60/g protein) and 3. 7 ± 0. 2 g/L (~39/g protein) at the 4-mL scale with oxygen supplementation. This provides an average 95% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nitrogenous bases and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of more than 20 different proteins. For example, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.
Olsen et al. (Thu,) studied this question.