| Autor: |
Dirk Weuster-Botz, Dominik Maslak, Matthias Freiherr von Roman, Alexander Vogt, Dariusch Hekmat, Peter Breitschwerdt, Sonja Berensmeier, Christoph Ströhle |
| Rok vydání: |
2015 |
| Předmět: |
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| Zdroj: |
Journal of Biotechnology. 194:84-90 |
| ISSN: |
0168-1656 |
| Popis: |
Recombinant enhanced green fluorescent protein (eGFP) is used as a marker in numerous applications in biomedical research and diagnostics. For these applications, the macromolecule needs to be provided in a highly purified form. The conventional purification process of eGFP usually consists of multiple subsequent preparative chromatography steps. Since this procedure is costly and time-consuming, an alternative chromatography-free purification process was investigated. This process was a combination of three-phase partitioning (TPP) and preparative crystallization including an ultrafiltration/diafiltration (UF/DF) intermediate step. After the TPP step, eGFP with a purity level suitable for preparative crystallization of 82.5–85.0% and a yield of 84–92% was obtained depending on the scale. After cross-flow UF/DF, the crystallization was performed in parallelized mL-scale stirred tanks. A favorable robust crystal morphology was obtained combined with fast crystallization kinetics when two polyethylenglycols and ethanol were used simultaneously as crystallization additives. The crystallization process can easily be scaled-up to obtain large amounts of highly purified, concentrated eGFP with a purity >99% after a crystal wash step and resolubilization. The proposed chromatography-free purification procedure gives reason to expect significant reductions of costs and required process time compared to conventional preparative chromatography. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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