title: Non-chromatographic preparative purification of enhanced green fluorescent protein.
abstract: 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.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany. Electronic address: hekmat@lrz.tum.de.
authors.forename: Dariusch
authors.initials: D
authors.lastname: Hekmat
authors.affiliation: Research Center for Industrial Biotechnology, Technische Universität München, Boltzmannstr. 17, 85748 Garching, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Maslak
authors.affiliation: Bioseparation Engineering Group, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Freiherr von Roman
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Breitschwerdt
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Ströhle
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Vogt
authors.affiliation: Bioseparation Engineering Group, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Berensmeier
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
mesh_terms: D049452:Green Fluorescent Proteins / Q000302:isolation & purification*; D014462:Ultrafiltration / Q000379:methods
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Clarified homogenized fermentation broth; Enhanced green fluorescent protein; Preparative crystallization; Protein purification; Stirred-tank crystallizer