Aqueous Biphasic Systems Composed of Cholinium Chloride and Polymers as Effective Platforms for the Purification of Recombinant Green Fluorescent Protein


Green fluorescent protein (GFP) has excellent properties as a biosensor and biomarker; however, its widespread use is limited by its purification costs. Alternative low-cost purification techniques can overcome this issue. The aim of this work was to evaluate aqueous biphasic systems (ABS) composed of cholinium chloride ([Ch]Cl) and different polymers as effective platforms to recover GFP from cell lysate of recombinant Escherichia coli BL21. All systems completely extracted GFP from cell lysate (>99%) into the polymeric- or [Ch]Cl-rich phases. In general, [Ch]Cl-based ABS allowed a good purification capacity (GFP 80-100% pure), with the best results (approximately 100% pure GFP) achieved with a polypropylene glycol (PPG)-400/[Ch]Cl ABS in a single-step extraction or in a two-step extraction (back-extraction) by the integration of a polyethylene glycol (PEG)/sodium polyacrylate+[Ch]Cl ABS with a following stage using a PEG/[Ch]Cl-based ABS. Additionally, to demonstrate the potential of the PPG-400/[Ch]Cl ABS in downstream processing, solvent recycling and GFP polishing were carried out using ultrafiltration. Finally, the capacity of the PPG-400/[Ch]Cl ABS to extract other fluorescent proteins was also confirmed. The results clearly demonstrated that the PPG-400/[Ch]Cl ABS can be applied in downstream processing for the purification of proteins, not only enhancing purification yields but also providing simpler, quicker, cost-effective, and biocompatible processes.



subject category

Chemistry; Science & Technology - Other Topics; Engineering


dos Santos, NV; Martins, M; Santos-Ebinuma, VC; Ventura, SPM; Coutinho, JAP; Valentini, SR; Pereira, JFB

our authors


This work was developed within the scope of the project "Optimization and Scale-up of Novel Ionic-Liquid-based Purification Processes for Recombinant Green Fluorescent Protein produced by Escherichia coli", process 2014/19793-3, cofunded by FAPESP (Sao Paulo Research Foundation, Brazil) and FCT (Portuguese Foundation for Science and Technology, Portugal). N.V.S. and J.F.B.P. acknowledge financial support from FAPESP through the projects 2016/07529-5 and 2014/16424-7, respectively. This work was also developed within the scope of the project CICECO Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT ref. UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate cofinanced by FEDER under the PT2020 Partnership Agreement. M.M. thanks FCT for the doctoral grant SFRH/BD/122220/2016. S.P.M.V. acknowledges the IF contract IF/00402/2015. The authors also acknowledge the support from CNPq (National Council for Scientific and Technological Development, Brazil) and CAPES (Coordination of Superior Level Staff Improvement, Brazil). The authors are also grateful for Prof. Danielle Biscaro Pedrolli for kindly providing YFP and RFP samples.

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