Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, 14476, Potsdam, Germany.
Faculty of Health Sciences, Joint Faculty of the Brandenburg University of Technology Cottbus-Senftenberg, The Brandenburg Medical School Theodor Fontane and the University of Potsdam, Potsdam, Germany.
BioDrugs. 2020 Jun;34(3):327-348. doi: 10.1007/s40259-020-00417-y.
Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development.
蛋白质是药物靶点的主要来源，其中一些本身就具有治疗潜力。在这些蛋白质中，膜蛋白约占主要药物靶点的 50%。在药物发现的过程中，开发出能够以简单的方式高质量生产不同类型蛋白质的快速方法，对于结构和功能分析非常重要。由于无细胞膜限制，无细胞体系作为一种有吸引力的替代方法，正在逐渐兴起，用于蛋白质的生产。在生物生产方面，基于不同来源的细胞裂解物的开放式体系，具有批次间一致性，为目标蛋白质的无细胞合成起到了催化剂的作用。最重要的是，蛋白质可以进行下游应用处理，如纯化和功能分析，而无需转染、选择和克隆的扩展。在过去的 5 年中，已经有越来越多的新型无细胞裂解物来源于多种生物体，并且它们被用于合成各种不同的蛋白质。尽管取得了这些进展，但在可扩展性、成本效益、蛋白质折叠和功能方面仍然存在重大挑战。在这篇综述中，我们介绍了不同来源的无细胞体系的概述及其在广泛的蛋白质生产中的应用。此外，本文还讨论了源自中国仓鼠卵巢和 Sf21 裂解物的无细胞体系的一些最新进展，这些体系包含内源性有效的转位活跃微粒体，用于合成膜蛋白。我们特别强调了内部核糖体进入位点序列的使用，以更有效地进行蛋白质生产，以及非典型氨基酸的定点掺入在标记应用和使用无细胞体系创建抗体药物偶联物方面的重要性。我们还讨论了克服涉及将无细胞平台从实验室水平商业化用于未来药物开发的主要挑战的策略。
