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首页> 外文期刊>Biotechnology Progress >A Simplified and Robust Protocol for Immunoglobulin Expression in Escherichia coli Cell-Free Protein Synthesis Systems
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A Simplified and Robust Protocol for Immunoglobulin Expression in Escherichia coli Cell-Free Protein Synthesis Systems

机译：免疫球蛋白在大肠杆菌无细胞蛋白合成系统中表达的简化和鲁棒协议。

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Cell-free protein synthesis (CFPS) systems allow for robust protein expression with easy manipulation of conditions to improve protein yield and folding. Recent technological developments have significantly increased the productivity and reduced the operating costs of CFPS systems, such that they can compete with conventional in vivo protein production platforms, while also offering new routes for the discovery and production of biotherapeutics. As cell-free systems have evolved, productivity increases have commonly been obtained by addition of components to previously designed reaction mixtures without careful reexamination of the essentiality of reagents from previous generations. Here we present a systematic sensitivity analysis of the components in a conventional Escherichia coli CFPS reaction mixture to evaluate their optimal concentrations for production of the immunoglobulin G trastuzumab. We identify eight changes to the system, which result in optimal expression of trastuzumab. We find that doubling the potassium glutamate concentration, while entirely eliminating pyruvate, coenzyme A, NAD, total tRNA, folinic acid, putrescine and ammonium glutamate, results in a highly productive cell-free system with a 95% reduction in reagent costs (excluding cell-extract, plasmid, and T7 RNA polymerase made in-house). A larger panel of other proteins was also tested and all show equivalent or improved yields with our simplified system. Furthermore, we demonstrate that all of the reagents for CFPS can be combined in a single freeze-thaw stable master mix to improve reliability and ease of use. These improvements are important for the application of the CFPS system in fields such as protein engineering, high-throughput screening, and biotherapeutics. (C) 2015 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers
机译：无细胞蛋白质合成（CFPS）系统可通过轻松控制条件来提高蛋白质表达水平，从而提高蛋白质产量和折叠能力。最近的技术发展显着提高了CFPS系统的生产率并降低了其运行成本，因此它们可以与传统的体内蛋白质生产平台竞争，同时也为生物治疗剂的发现和生产提供了新途径。随着无细胞系统的发展，通常在不仔细检查前几代试剂的本质的情况下，通过将组分添加到先前设计的反应混合物中来提高生产率。在这里，我们介绍了常规大肠杆菌CFPS反应混合物中各成分的系统敏感性分析，以评估其产生免疫球蛋白G曲妥珠单抗的最佳浓度。我们确定了系统的八个变化，这导致曲妥珠单抗的最佳表达。我们发现，将谷氨酸钾浓度加倍，同时完全消除丙酮酸，辅酶A，NAD，总tRNA，亚叶酸，腐胺和谷氨酸铵，可实现高效无细胞系统，试剂成本降低95％（不包括细胞） -提取，质粒和T7 RNA聚合酶内部制作）。还测试了更大范围的其他蛋白质，并且使用我们简化的系统，所有蛋白质均显示出相同或更高的产量。此外，我们证明了CFPS的所有试剂都可以在单一的冻融稳定的预混物中混合使用，以提高可靠性和易用性。这些改进对于CFPS系统在蛋白质工程，高通量筛选和生物治疗等领域的应用至关重要。 （C）2015年Wiley Periodicals，Inc.代表美国化学工程师学会出版的《作者生物技术进展》

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