Levine Max Z, Gregorio Nicole E, Jewett Michael C, Watts Katharine R, Oza Javin P
Department of Biological Sciences, California Polytechnic State University, San Luis Obispo; Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo.
Center for Applications in Biotechnology, California Polytechnic State University, San Luis Obispo; Department of Chemistry and Biochemistry, California Polytechnic State University.
J Vis Exp. 2019 Feb 25(144). doi: 10.3791/58882.
Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.
在过去的50年里，无细胞蛋白质合成（CFPS）已成为一项强大的技术，可在试管内利用细胞的转录和翻译能力。通过无需维持细胞的活力，并消除细胞屏障，CFPS已成为传统上具有挑战性的蛋白质生物制造新兴应用、代谢工程快速原型制作以及功能基因组学应用的基础。我们实施基于大肠杆菌的CFPS平台的方法使新用户能够使用许多这些应用。在这里，我们描述了通过使用富集培养基、 baffled烧瓶以及基于可调谐超声处理的可重复细胞裂解方法来制备提取物的方法。如果事先准备好适当的试剂储备，那么从实验设置到数据分析，这种提取物可用于蛋白质表达，仅需5小时就能产生900μg/mL或更多的超级折叠绿色荧光蛋白（sfGFP）。获得试剂的估计启动成本为4500美元，这将维持数千次反应，估计成本为每微克产生的蛋白质0.021美元或每微升反应0.019美元。此外，由于试剂预混物的优化，蛋白质表达方法反映了市售系统中反应设置的简便性，而成本仅为其一小部分。为了使用户能够利用CFPS平台的灵活性进行广泛应用，我们已经确定了该平台的各个方面，可根据可用资源和所需的蛋白质表达结果进行调整和优化。
