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Non-chromatographic preparative purification of enhanced green fluorescent protein

Hekmat, Dariusch, Maslak, Dominik, Freiherr von Roman, Matthias, Breitschwerdt, Peter, Ströhle, Christoph, Vogt, Alexander, Berensmeier, Sonja, Weuster-Botz, Dirk
Journal of biotechnology 2015 v.194 pp. 84-90
additives, biomedical research, chromatography, crystallization, diagnostic techniques, ethanol, green fluorescent protein, purification methods, tanks, ultrafiltration
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.