Aqueous Biphasic Systems Composed of Cholinium Chloride and Polymers as Effective Platforms for the Purification of Recombinant Green Fluorescent Protein
authors dos Santos, NV; Martins, M; Santos-Ebinuma, VC; Ventura, SPM; Coutinho, JAP; Valentini, SR; Pereira, JFB
nationality International
journal ACS SUSTAINABLE CHEMISTRY & ENGINEERING
author keywords Aqueous biphasic systems; Cholinium chloride; Green fluorescent protein; Purification; Integrated downstream process
keywords MOLECULARLY IMPRINTED POLYMERS; METAL AFFINITY-CHROMATOGRAPHY; ESCHERICHIA-COLI HOMOGENATE; SOLID-PHASE EXTRACTION; 2-PHASE SYSTEMS; IONIC LIQUIDS; POLYETHYLENE-GLYCOL; RAPID PURIFICATION; SEPARATION; YIELD
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]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.
publisher AMER CHEMICAL SOC
issn 2168-0485
year published 2018
volume 6
issue 7
beginning page 9383
ending page 9393
digital object identifier (doi) 10.1021/acssuschemeng.8b01730
web of science category Chemistry, Multidisciplinary; GREEN & SUSTAINABLE SCIENCE & TECHNOLOGY; Engineering, Chemical
subject category Chemistry; Science & Technology - Other Topics; Engineering
unique article identifier WOS:000444924500143
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journal impact factor 6.140
5 year journal impact factor 6.415
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