In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based Protection of LET) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

中文翻译：

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通过使用DNA结合蛋白从大肠杆菌和纳氏弧菌提取物中的线性模板提高无细胞基因表达的产量

在基于粗提物的无细胞蛋白质合成（CFPS）中，DNA模板被转录并翻译为功能性蛋白质。尽管线性表达模板（LETs）较省力且生成昂贵，但由于增加的稳定性和对反应提取物中存在的核酸外切酶的保护作用，通常需要质粒模板优于PCR生成的LETs。在这里，我们证明了将dsDNA结合蛋白添加到CFPS反应中，称为单链Cro蛋白（scCro），可实现LET的末端保护。此CroP-LET（基于scCro的LET保护）方法可将大肠杆菌CFPS反应中的LET中的sfGFP表达水平有效提高6倍。我们的产量可与其他提供化学和酶促DNA稳定作用的策略相媲美。大肠杆菌CFPS。值得注意的是，我们还报告了CroP-LET方法成功地提高了非模型生物衍生的CFPS平台的产量。我们的结果表明，CroP-LET在天然弧菌CFPS平台中使sfGFP产量增加了18倍。随着CFPS平台的快速扩展应用，该方法提供了一种实用且可通用的解决方案来保护线性表达DNA模板。