Cell‐free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just‐add‐water cell‐free system

Biotherapeutics have many promising applications, such as anti‐cancer treatments, immune suppression, and vaccines. However, due to their biological nature, some biotherapeutics can be challenging to rapidly express and screen for activity through traditional recombinant methods. For example, difficult‐to‐express proteins may be cytotoxic or form inclusion bodies during expression, increasing the time, labor, and difficulty of purification and downstream characterization. One potential pathway to simplify the expression and screening of such therapeutics is to utilize cell‐free protein synthesis. Cell‐free systems offer a compelling alternative to in vivo production, due to their open and malleable reaction environments. In this work, we demonstrate the use of cell‐free systems for the expression and direct screening of the difficult‐to‐express cytotoxic protein onconase. Using cell‐free systems, onconase can be rapidly expressed in soluble, active form. Furthermore, the open nature of the reaction environment allows for direct and immediate downstream characterization without the need of purification. Also, we report the ability of a ”just‐add‐water“ lyophilized cell‐fee system to produce onconase. This lyophilized system remains viable after being stored above freezing for up to one year. The beneficial features of these cell‐free systems make them compelling candidates for future biotherapeutic screening and production.