Purification of green fluorescent protein using fast centrifugal partition chromatography


The green fluorescent protein (GFP) is a biomolecule used in many biological applications such as biomarkers and biosensors, which require high purity levels. It is usually obtained from recombinant Escherichia coli strains, which also produces other endogenous proteins, demanding multiple purification steps, and consequently, increasing the overall costs to obtain pure GFP. Simpler and cheaper purification methods like Aqueous Biphasic Systems (ABS) were already successfully applied to purify GFP at lab scale. Therefore, the development of automatized industrially compatible purification platforms, such as countercurrent chromatography using ABS, can potentially improve the GFP production. This work studied the continuous purification of the variant enhanced GFP (EGFP) by applying ABS composed of polyethylene glycol (PEG 8000), sodium polyacrylate (NaPA 8000) and sodium sulfate (Na2SO4) as electrolyte. An initial screening was carried by changing the electrolyte content in the ABS. The increase of this condition has demonstrated an increase on the EGFP partition for the PEG-rich phase. The most efficient ABS and, at the same time, with the most appropriate conditions, namely the system composed of 15 wt% PEG 8000 + 4.5 wt% NaPA 8000 + 2.5 wt% Na2SO4 was chosen and applied on the fast centrifugal partition chromatography (FCPC). After optimization, the best operational conditions were identified, i.e. a flow rate of 2.5 mL.min(-1) and rotation speed of 2000 rpm at ascending mode, and the best results obtained, meaning a purification of 89.93% and a recovery yield of 82.3%, confirming the potential of FCPC to the continuous purification of EGFP.



subject category

Engineering, Chemical


Soares, BP; Santos, JHPM; Martins, M; Almeida, MR; Santos, NV; Freire, MG; Santos-Ebinuma, VC; Coutinho, JAP; Pereira, JFB; Ventura, SPM

our authors


This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. The authors are also grateful to FCT by the financial support considering the Project Optimization and Scale-up of Novel Ionic Liquidbased Purification Processes for Recombinant Green Fluorescent Protein produced by Escherichia coli, reference FAPESP/19793/2014. J.H.P.M. Santos acknowledge FAPESP grant 2018/25994-2. The authors also thank FCT for the doctoral grants SFRH/BD/122220/2016 of M. Martins. S.P.M. Ventura acknowledges for the IF contract IF/00402/2015. J.F.B. Pereira and N. V. dos Santos acknowledges financial support from FAPESP through the project 2014/16424-7 and 2016/07529-5, respectively.

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