Zarina Levitskaya, Zheng Ser, Hiromi Koh, Wang Shi Mei, Sharon Chee, Radoslaw Mikolaj Sobota and John F. Ghadessy
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引用次数: 0
摘要
表型筛选是了解和设计复杂生物系统的重要工具。我们在无细胞蛋白质合成(CFPS)技术的开发中展示了这种方法的功能。表型筛选发现了许多能在酵母裂解物 CFPS 反应中提高蛋白质产量的化合物。值得注意的是,其中许多化合物都是竞争性 ATP 激酶抑制剂,利用其固有的底物杂合性将 ATP 通量重新导向异源蛋白表达。以化学蛋白组学为指导的菌株工程学部分表征了药物效应,在删除 HSP70 合子中消耗 ATP 的 SSA1 成分后,蛋白质产量增加了 30%。此外,药物介导的新陈代谢改组与模板优化相结合,利用迄今为止效率较低但成本效益更高的葡萄糖能量再生系统,在酵母 CFPS 中产生了迄今为止最高的蛋白质产量。我们的方法凸显了靶标识别表型筛选和靶标鉴定在消除细胞裂解物复杂性方面的效用,为不断扩大的 CFPS 改进策略范围增添了新的内容。
Engineering cell-free systems by chemoproteomic-assisted phenotypic screening†
Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.