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

Autor(es)

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]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: ACS SUSTAINABLE CHEMISTRY & ENGINEERING; v. 6, n. 7, p. 9383-9393, JUL 2018.
• Citações Web of Science: 6

Resumo

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)

• Processo FAPESP: 14/16424-7 - Otimização e aumento de escala do processo de extração líquido-líquido com líquidos iônicos (LIs) como ferramenta de separação sustentável do biofármaco antileucêmico L-asparaginase (ASPase)
• Beneficiário: Jorge Pereira
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 14/19793-3 - Otimização e aumento de escala de processos com líquidos iônicos como nova ferramenta de purificação da proteína verde fluorescente recombinante produzida por Escherichia coli "GFPurIL"
• Beneficiário: Sandro Roberto Valentini
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 16/07529-5 - Desenvolvimento de processos de purificação líquido-líquido e incorporação em matriz polimérica nanoestruturada da proteína verde fluorescente recombinante produzida por Escherichia coli
• Beneficiário: Nathalia Vieira Porphirio Veríssimo
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto