Purification of green fluorescent protein using fast centrifugal partition chromatography

Author(s)

Soares, Bruna P. ^[1] ; Santos, Joao H. P. M. ^{[2, 1]} ; Martins, Margarida ^[1] ; Almeida, Mafalda R. ^[1] ; Santos, V, Nathalia ; Freire, Mara G. ^[1] ; Santos-Ebinuma, Valeria C. ^[3] ; Coutinho, Joao A. P. ^[1] ; Pereira, Jorge F. B. ^{[3, 4]} ; Ventura, Sonia P. M. ^[1]

Affiliation

^[1] Univ Aveiro, CICECO Aveiro Inst Mat, Dept Chem, P-3810193 Aveiro - Portugal

^[2] Univ Sao Paulo, Dept Biochem & Pharmaceut Technol, BR-05508000 Sao Paulo - Brazil

^[3] Santos, Nathalia, V, Sao Paulo State Univ, Sch Pharmaceut Sci, Dept Bioproc & Biotechnol, UNESP, BR-14800903 Araraquara, SP - Brazil

^[4] Univ Coimbra, Dept Chem Engn, CIEPQPF, Rua Silvio Lima, Polo 2 Pinhal de Marrocos, P-3030790 Coimbra - Portugal

• Document type: Journal article
• Source: Separation and Purification Technology; v. 257, FEB 15 2021.
• Web of Science Citations: 2

Abstract

The green fluorescent protein (GFP) is a biomolecule used in many biological applications such as biomarkers and biosensors, which require high purity levels. It is usually obtained from recombinant Escherichia coli strains, which also produces other endogenous proteins, demanding multiple purification steps, and consequently, increasing the overall costs to obtain pure GFP. Simpler and cheaper purification methods like Aqueous Biphasic Systems (ABS) were already successfully applied to purify GFP at lab scale. Therefore, the development of automatized industrially compatible purification platforms, such as countercurrent chromatography using ABS, can potentially improve the GFP production. This work studied the continuous purification of the variant enhanced GFP (EGFP) by applying ABS composed of polyethylene glycol (PEG 8000), sodium polyacrylate (NaPA 8000) and sodium sulfate (Na2SO4) as electrolyte. An initial screening was carried by changing the electrolyte content in the ABS. The increase of this condition has demonstrated an increase on the EGFP partition for the PEG-rich phase. The most efficient ABS and, at the same time, with the most appropriate conditions, namely the system composed of 15 wt% PEG 8000 + 4.5 wt% NaPA 8000 + 2.5 wt% Na2SO4 was chosen and applied on the fast centrifugal partition chromatography (FCPC). After optimization, the best operational conditions were identified, i.e. a flow rate of 2.5 mL.min(-1) and rotation speed of 2000 rpm at ascending mode, and the best results obtained, meaning a purification of 89.93% and a recovery yield of 82.3%, confirming the potential of FCPC to the continuous purification of EGFP. (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: 18/25994-2 - Development of novel platforms for PEGylation of proteins with therapeutic potential using microfluidics
• Grantee: João Henrique Picado Madalena Santos
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• 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)