School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive NW, Atlanta, Georgia 30332-0100, United States.
ACS Synth Biol. 2023 Oct 20;12(10):3131-3136. doi: 10.1021/acssynbio.3c00312. Epub 2023 Sep 19.
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 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.
细胞裂解——例如通过超声处理或珠磨处理——是为无细胞表达系统制备提取物的关键步骤。为了创建具有高蛋白质生产能力的提取物，标准做法是充分裂解细胞，彻底破坏细胞膜，从而提取表达机制，但不降解该机制。在这里，我们研究了不同超声能量输入对提取物蛋白质生产能力的影响。虽然存在特定于操作子的最佳超声能量输入是众所周知的，但我们的研究结果表明，从给定表达模板获得最大蛋白质产量的超声能量输入可能取决于质粒浓度、转录和翻译特征（例如启动子）以及其他表达载体成分（例如复制起点）。这些结果表明，超声处理方案不能标准化为单一最佳方案，提出了提高蛋白质产量的策略，并更广泛地强调了需要更好的细胞提取物特性描述的指标和方案。
