ABSTRACT Cell‐free gene expression systems offer cell‐like functionalities outside the confines of the cell, garnering increasing interest for applications from biomanufacturing to sensing. As applications expand, the need to implement economically scaled processes to produce cellular lysates grows. The protocols to produce these cellular lysates are complex, and the impact of altering many of the process variables remains understudied. Here, we set out to evaluate the effect of extended incubations at several points in the extract preparation process with the goal of identifying breakpoints that would enable flexibility in process implementation. As a model, we prepared lysates from 50 L cultures instead of typical 1 L volumes. We produced 72 lysates, 36 that were incubated overnight before and after culture centrifugation, and 36 that were incubated with and without a run‐off reaction, each across different temperatures. We found that incubations before and after culture centrifugation substantially increased variability between culture replicates but did not reduce cell‐free protein synthesis activity, contrary to conventional wisdom that materials should be kept cold as much as possible throughout the process. We also observed that omitting the run‐off reaction reduced yields but resulted in lysates that were robust to incubation up to room temperature overnight. When a run‐off reaction was included, activity dropped both as a function of duration and temperature, and the overall variability increased. Our work offers potential options for flexibility in implementing lysate production processes and motivates further investigation into how key processing steps relate to cell‐free expression activity.
Rhea et al. (2026) studied this question.