Abstract：
Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.
摘要：
无细胞基因表达系统用于许多应用中，包括药物制造，诊断,和教育工具包。这些系统中的非荧光蛋白的准确定量仍然是一个挑战。为了应对这一挑战，我们报道了优化的四半胱氨酸小螺旋作为融合蛋白和FlasH染料的独立报道分子的适应和使用.荧光报告螺旋短到足以在引物对上编码以通过PCR标记任何感兴趣的蛋白质。标记的蛋白质和独立的报道分子都可以用标准的96/384孔板读数器实时或在无细胞表达反应结束时定量检测，RT-qPCR系统，或凝胶电泳无需染色。荧光信号稳定，与蛋白质浓度呈线性关系，实现产品量化。我们修改了报告基因以研究无细胞表达动力学和工程核糖体活性。我们预计荧光小螺旋报告基因将有助于体外转录和翻译系统的工程改造。
