High-throughput preparation methods of crude extract for robust cell-free protein synthesis.
High-throughput preparation methods of crude extract for robust cell-free protein synthesis.
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DOI：
10.1038/srep08663
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发表时间：
2015-03-02
影响因子：
4.6
通讯作者：
Jewett MC
中科院分区：
文献类型：
--
作者：
Kwon YC;Jewett MC
基于粗提物的无细胞蛋白质合成（CFPS）已成为高通量蛋白质生产和遗传部分表征的强大技术平台。不幸的是，高活性提取物的稳健制备通常需要专门且昂贵的设备，并且可能是劳动和时间密集型的。此外，细胞裂解程序可能难以标准化，导致不同实验室的提取物性能不同。这些挑战限制了该领域的新进入者和新的应用，例如提高提取性能的综合基因组工程计划。为了应对这些挑战，我们开发了一种基于超声处理的CFPS通用且易于获得的高通量粗提物制备方法。为了验证我们的方法，我们研究了两种大肠杆菌菌株：BL 21星星™（DE 3）和K12 MG 1655变体，通过仅改变几个参数实现了相似的生产率（定义为CFPS产率，以g/L计）。此外，我们观察到从跨越三个数量级的培养体积（10 mL培养管至10 L发酵）产生的细胞提取物的相同生产率。 我们预计，我们的快速和强大的提取物制备方法将加快筛选基因组工程菌株的CFPS应用，使非模式生物的高活性提取物成为可能，并促进CFPS在合成生物学和生物技术中的更广泛使用。
Crude extract based cell-free protein synthesis (CFPS) has emerged as a powerful technology platform for high-throughput protein production and genetic part characterization. Unfortunately, robust preparation of highly active extracts generally requires specialized and costly equipment and can be labor and time intensive. Moreover, cell lysis procedures can be hard to standardize, leading to different extract performance across laboratories. These challenges limit new entrants to the field and new applications, such as comprehensive genome engineering programs to improve extract performance. To address these challenges, we developed a generalizable and easily accessible high-throughput crude extract preparation method for CFPS based on sonication. To validate our approach, we investigated two Escherichia coli strains: BL21 Star™ (DE3) and a K12 MG1655 variant, achieving similar productivity (defined as CFPS yield in g/L) by varying only a few parameters. In addition, we observed identical productivity of cell extracts generated from culture volumes spanning three orders of magnitude (10 mL culture tubes to 10 L fermentation). We anticipate that our rapid and robust extract preparation method will speed-up screening of genomically engineered strains for CFPS applications, make possible highly active extracts from non-model organisms, and promote a more general use of CFPS in synthetic biology and biotechnology.
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影响因子：
4.1
作者：
Bundy, Bradley C.;Swartz, James R.
通讯作者：
Swartz, James R.
影响因子：
20.3
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影响因子：
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影响因子：
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通讯作者：
Jewett MC