Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0314, USA.
Sci Rep. 2018 Feb 22;8(1):3488. doi: 10.1038/s41598-018-21739-6.
Protein degradation is a fundamental process in all living cells and is essential to remove both damaged proteins and intact proteins that are no longer needed by the cell. We are interested in creating synthetic genetic circuits that function in a cell-free expression system. This will require not only an efficient protein expression platform but also a robust protein degradation system in cell extract. Therefore, we purified and tested the activity of E. coli ClpXP protease in cell-free transcription-translation (TX-TL) systems that used E. coli S30 cell extract. Surprisingly, our studies showed that purified ClpXP added to the TX-TL system has very low proteolytic activity. The low activity of ClpXP was correlated with the rapid consumption of adenosine triphosphate (ATP) in cell extract. We improved the activity of ClpXP in cell extract by adding exogenous ATP and an energy regeneration system. We then established conditions for both protein synthesis, and protein degradation by ClpXP to occur simultaneously in the TX-TL systems. The optimized conditions for ClpXP activity will be useful for creating tunable synthetic genetic circuits and in vitro synthetic biology.
蛋白质降解是所有活细胞中的一个基本过程，对于去除受损蛋白质和细胞不再需要的完整蛋白质至关重要。我们有兴趣创建在无细胞表达系统中起作用的合成遗传回路。这不仅需要高效的蛋白质表达平台，还需要细胞提取物中的稳健蛋白质降解系统。因此，我们在使用大肠杆菌 S30 细胞提取物的无细胞转录-翻译 (TX-TL) 系统中纯化并测试了大肠杆菌 ClpXP 蛋白酶的活性。令人惊讶的是，我们的研究表明，添加到 TX-TL 系统中的纯化 ClpXP 具有非常低的蛋白水解活性。ClpXP 的低活性与细胞提取物中三磷酸腺苷 (ATP) 的快速消耗有关。我们通过添加外源 ATP 和能量再生系统来提高细胞提取物中 ClpXP 的活性。然后，我们建立了在 TX-TL 系统中同时进行蛋白质合成和 ClpXP 蛋白降解的条件。优化的 ClpXP 活性条件将有助于创建可调谐的合成遗传回路和体外合成生物学。
