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.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Hekmat D,Maslak D,Freiherr von Roman M,Breitschwerdt P,Ströhle C,Vogt A,Berensmeier S,Weuster-Botz Ddoi
10.1016/j.jbiotec.2014.11.027subject
Has Abstractpub_date
2015-01-20 00:00:00pages
84-90eissn
0168-1656issn
1873-4863pii
S0168-1656(14)01025-6journal_volume
194pub_type
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