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首页> 外文期刊>Journal of Biotechnology >Integrated development of up- and downstream processes supported by the Cherry-Tag (TM) for real-time tracking of stability and solubility of proteins
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Integrated development of up- and downstream processes supported by the Cherry-Tag (TM) for real-time tracking of stability and solubility of proteins

机译：Cherry-Tag（TM）支持的上下游工艺的集成开发，可实时跟踪蛋白质的稳定性和溶解性

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Product analytics is the bottleneck of most processes in bioprocess engineering, as it is rather time-consuming. Real-time and in-line product tracing without sample pre-treatment is only possible for few products. The Cherry-Tag (TM) (Delphi Genetics, Belgium) which can be fused to any target protein allows for straightforward product analytics by VIS absorption measurements. When the fused protein becomes unstable or insoluble, the chromophore function of the group is lost, which makes this technology an ideal screening tool for solubility and stability in up- and downstream process development. The Cherry-Tag (TM) technology will be presented for the tagged enzyme glutathione-S-transferase (GST) from Escherichia coli in a combined up- and downstream process development study. High-throughput cultivations were carried out in a 48-well format in a BioLector system (m2p-Labs, Germany). The best cultivation setup of highest product titer was scaled up to a 2.5 L shake flask culture, followed by a selective affinity chromatography product capturing step. In upstream applications the tag was capable of identifying conditions where insoluble and non-native inclusion bodies were formed. In downstream applications the red-colored product was found to be bound effectively to a GST affinity column. Thus, it was identified to be a native and active protein, as the binding mechanism relies on catalytic activity of the enzyme. The Cherry-Tag (TM) was found to be a reliable and quantitative tool for real-time tracking of stable and soluble proteins in up- and downstream processing applications. Denaturation and aggregation of the product can be detected in-line at any stage of the process. Critical stages can be identified and subsequently changed or replaced. (C) 2015 Elsevier B.V. All rights reserved.
机译：产品分析是生物过程工程中大多数过程的瓶颈，因为它相当耗时。未经样品预处理的实时和在线产品跟踪仅适用于少数产品。可与任何靶蛋白融合的Cherry-Tag（TM）（比利时德尔斐遗传学）可通过VIS吸收测量进行直接的产品分析。当融合蛋白变得不稳定或不溶时，该基团的发色团功能丧失，这使该技术成为上下游工艺开发中溶解度和稳定性的理想筛选工具。 Cherry-Tag（TM）技术将在上下游联合开发研究中针对来自大肠杆菌的标记酶谷胱甘肽S-转移酶（GST）推出。在BioLector系统（m2p-Labs，德国）中以48孔格式进行高通量培养。将最高产品滴度的最佳培养设置扩大到2.5 L摇瓶培养，然后进行选择性亲和层析产品捕获步骤。在上游应用中，标签能够识别形成不溶性和非天然包涵体的条件。在下游应用中，发现红色产品与GST亲和柱有效结合。因此，由于结合机理依赖于酶的催化活性，因此被鉴定为天然和活性蛋白。发现Cherry-Tag（TM）是一种可靠的定量工具，用于实时跟踪上下游加工应用中的稳定和可溶性蛋白质。产品的变性和聚集可以在过程的任何阶段进行在线检测。关键阶段可以被识别，随后可以更改或替换。 （C）2015 Elsevier B.V.保留所有权利。

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