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

Author(s)

dos Santos, Nathalia V. ^[1] ; Martins, Margarida ^[2] ; Santos-Ebinuma, Valeria C. ^[1] ; Ventura, Sonia P. M. ^[2] ; Coutinho, Joao A. P. ^[2] ; Valentini, Sandro R. ^[3] ; Pereira, Jorge F. B. ^[1]

Affiliation

^[1] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, Rodovia Araraquara Jau Km 01, BR-14800903 Araraquara, SP - Brazil

^[2] Univ Aveiro, Dept Chem, CICECO Aveiro Inst Mat, P-3810193 Aveiro - Portugal

^[3] Sao Paulo State Univ UNESP, Sch Pharmaceut Sci, Dept Biol Sci, Rodovia Araraquara Jau Km 01, BR-14800903 Araraquara, SP - Brazil

• Document type: Journal article
• Source: ACS SUSTAINABLE CHEMISTRY & ENGINEERING; v. 6, n. 7, p. 9383-9393, JUL 2018.
• Web of Science Citations: 6

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. (AU)

• FAPESP's process: 14/16424-7 - Optimization and scale-up of liquid-liquid extraction process with ionic liquids (ILs) as a sustainable tool for the separation of the anti-leukemia biopharmaceutical L-asparaginase (ASPase)
• Grantee: Jorge Pereira
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 14/19793-3 - Optimization and scale-up of novel Ionic-Liquid-based purification processes for recombinant green fluorescent protein produced by Escherichia coli "GFPurIL"
• Grantee: Sandro Roberto Valentini
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/07529-5 - Development of liquid-liquid purification process and incorporation in nanostructured polymeric matrix of recombinant green fluorescent protein produced by Escherichia coli
• Grantee: Nathalia Vieira Porphirio Veríssimo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)