INRAe, AgroParisTech, Micalis Institute, Université Paris-Saclay.
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI); Medical Faculty, University of Würzburg.
J Vis Exp. 2022 Aug 9(186). doi: 10.3791/64236.
Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.
无细胞蛋白质合成（CFPS）由于其众多优点，最近在合成生物学领域变得非常流行。使用线性 DNA 模板进行 CFPS 将进一步使其技术发挥出全部潜力，通过消除克隆、转化和质粒提取等步骤，减少实验时间。线性 DNA 可以通过 PCR 快速且轻松地扩增，以获得高浓度的模板，从而避免潜在的体内表达毒性。然而，线性 DNA 模板会被细胞提取物中天然存在的外切核酸酶迅速降解。已经提出了几种策略来解决这个问题，例如添加核酸酶抑制剂或线性 DNA 末端的化学修饰以进行保护。所有这些策略都需要额外的时间和资源，并且尚未获得接近质粒水平的蛋白质表达。这里提出了一种替代策略的详细方案，用于使用线性 DNA 模板进行 CFPS。通过使用缺乏外切核酸酶的敲除细胞的细胞提取物，线性 DNA 模板在不进行任何末端修饰的情况下保持完整。我们介绍了通过超声裂解和缓冲液校准从大肠杆菌 BL21 Rosetta2 ΔrecBCD 菌株制备细胞裂解物的步骤，专门用于 Mg-谷氨酸（Mg-glu）和 K-谷氨酸（K-glu）的线性 DNA。该方法能够在大肠杆菌 CFPS 中实现与质粒 DNA 相当的蛋白质表达水平。
