Non-chromatographic preparative purification of enhanced green fluorescent protein

Dariusch Hekmat, Dominik Maslak, Matthias Freiherr von Roman, Peter Breitschwerdt, Christoph Ströhle, Alexander Vogt, Sonja Berensmeier, Dirk Weuster-Botz

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


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.

Original languageEnglish
Pages (from-to)84-90
Number of pages7
JournalJournal of Biotechnology
StatePublished - 1 Jan 2015


  • Clarified homogenized fermentation broth
  • Enhanced green fluorescent protein
  • Preparative crystallization
  • Protein purification
  • Stirred-tank crystallizer


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