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Title: Aqueous biphasic systems composed of cholinium chloride and polymers as effective platforms for the purification of recombinant green fluorescent protein
Author: dos Santos, Nathalia V.
Martins, Margarida
Santos-Ebinuma, Valéria C.
Ventura, Sónia P. M.
Coutinho, João A. P.
Valentini, Sandro R.
Pereira, Jorge F. B.
Keywords: Aqueous biphasic systems
Cholinium chloride
Green fluorescent protein
Integrated downstream process
Issue Date: 2018
Publisher: American Chemical Society
Abstract: 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]Clbased 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 (backextraction) 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.
Peer review: yes
DOI: 10.1021/acssuschemeng.8b01730
Appears in Collections:CICECO - Artigos

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