Complex Dependence of Escherichia coli-based Cell-Free Expression on Sonication Energy During Lysis

Cell lysis-by sonication or bead beating, for example-is a key step in preparing extracts for cell-free expression systems. To create high protein-production capacity extracts, standard practice is to lyse cells sufficiently to thoroughly disrupt the membrane and thus extract expression machinery but without degrading that machinery. Here, we investigate the impact of different sonication energy inputs on the protein-production capacity of Escherichia coli extracts. While the existence of operator-specific optimal sonication energy inputs is widely known, our findings show that the sonication energy input that yields maximal protein output from a given expression template may depend on plasmid concentration, transcriptional and translational features (e.g., promoter), and other expression vector components (e.g., origin of replication). These results indicate that sonication protocols cannot be standardized to a single optimum, suggest strategies for improving protein yields, and more broadly highlight the need for better metrics and protocols for characterizing cell extracts.