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Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins

Paper ID Volume ID Publish Year Pages File Format Full-Text
22918 43394 2015 11 PDF Available
Title
Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins
Abstract

•The Cherry-Tag™ is presented as a quantitative real-time and in-line product tracking tool which is not fluorescence based.•The tag allowed for distinction between insoluble/non-native proteins and soluble/native species shown in a glutathione-S-transferase (GST) case study.•In upstream processing applications the Cherry-Tag™ was capable of identifying process conditions of inclusion body formation.•In downstream processing applications it was shown that the colored GST fusion protein was catalytically active and could be bound effectively to a GST affinity column.•The Cherry-Tag™ was identified as a reliable and quantitative tool for real-time tracking of stable and soluble proteins during bioprocess development.

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™ (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™ 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™ 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.

Keywords
Cherry-Tag™; Product analytics; Native protein; Micro-cultivation; BioLector
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Integrated development of up- and downstream processes supported by the Cherry-Tag™ for real-time tracking of stability and solubility of proteins
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 200, 20 April 2015, Pages 27–37
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us