期刊
ACS SUSTAINABLE CHEMISTRY & ENGINEERING
卷 6, 期 7, 页码 9383-9393出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssuschemeng.8b01730
关键词
Aqueous biphasic systems; Cholinium chloride; Green fluorescent protein; Purification; Integrated downstream process
资金
- FAPESP (Sao Paulo Research Foundation, Brazil) [2014/19793-3]
- FCT (Portuguese Foundation for Science and Technology, Portugal) [2014/19793-3]
- FAPESP [2016/07529-5, 2014/16424-7]
- CICECO Aveiro Institute of Materials [POCI-01-0145-FEDER-007679]
- national funds through the FCT/MEC [UID/CTM/50011/2013]
- FEDER
- FCT [SFRH/BD/122220/2016]
- CNPq (National Council for Scientific and Technological Development, Brazil)
- CAPES (Coordination of Superior Level Staff Improvement, Brazil)
- [IF/00402/2015]
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.
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