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Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates

机译：无细胞非天然氨基酸与替代能源系统和线性表达模板的结合

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Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation.
机译：在蛋白质合成过程中非天然氨基酸（uAAs）的位点特异性掺入通过添加独特的残基化学来扩展蛋白质组学代码。该领域提供了独特的工具来改善药代动力学，癌症治疗，疫苗开发，蛋白质组学和蛋白质工程。通过uAA掺入预测蛋白质特征的能力有限，因此需要一种具有快速筛选潜力的低成本系统。基于大肠埃希氏菌的无细胞蛋白质合成是uAA引入的引人注目的平台，这是因为反应环境的开放性和可及性。然而，典型的无细胞系统由于给试剂赋能的高成本而可能是昂贵的。通过使用替代能源，我们将uAA掺入CFPS的成本降低了55％。尽管替代能源系统降低了成本，但开发基因库的时间投入仍然很麻烦。无细胞系统允许直接使用称为线性表达模板的PCR产物，从而减轻了繁琐的质粒文库制备步骤。我们报告与uAA合并CFPS的具体成本，证明LETs与uAA合并合适的表达模板，并考虑使用基于LET的CFPS uAA合并表达降低劳动强度。

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