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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：综合开发樱桃标签（TM）支持的上下游流程，以实时跟踪蛋白质的稳定性和溶解度

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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。没有样品预处理的实时和在线产品追踪只有很少的产品。可以融合到任何靶蛋白的樱桃标签（TM）（Delphi Genetics，比利时）通过Vis吸收测量来允许直接的产品分析。当融合蛋白变得不稳定或不溶于溶解时，该组的发色团功能损失，这使得这项技术成为上游工艺发育中的溶解性和稳定性的理想筛选工具。樱桃标签（TM）技术将以来自大肠杆菌的标记酶谷胱甘肽-S-转移酶（GST）呈现出来自大肠杆菌的组合和下游工艺发育研究。在Biolper系统（M2P-Labs，Germany）中以48孔格式进行高通量培养。最高产物滴度的最佳培养设置被缩放到2.5L摇瓶培养，然后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夹杂物体的条件。在下游应用中，发现红彩色产物有效地绑定到GST亲和柱。因此，随着结合机制依赖于天然和活性蛋白质，因为结合机制依赖于酶的催化活性。发现樱桃标签（TM）是一种可靠的和定量工具，用于在上游和下游加工应用中实时跟踪稳定和可溶性蛋白质。可以在该方法的任何阶段在线检测产品的变性和聚集。可以识别关键阶段并随后更改或更换。 （c）2015 Elsevier B.v.保留所有权利。

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