Purification of the Recombinant Green Fluorescent Protein Using Aqueous Two-Phase System Composed of Recyclable CO2-Based Alkyl Carbamate Ionic Liquid

Abstract

The formation of aqueous two-phase system (ATPS) with the environmentally friendly and recyclable ionic liquid has been gaining popularity in the field of protein separation. In this study, the ATPSs comprising N,N-dimethylammonium N′,N′-dimethylcarbamate (DIMCARB) and thermo-responsive poly(propylene) glycol (PPG) were applied for the recovery of recombinant green fluorescent protein (GFP) derived from Escherichia coli. The partition behavior of GFP in the PPG + DIMCARB + water system was investigated systematically by varying the molecular weight of PPG and the total composition of ATPS. Overall, GFP was found to be preferentially partitioned to the hydrophilic DIMCARB-rich phase. An ATPS composed of 42% (w/w) PPG 1000 and 4.4% (w/w) DIMCARB gave the optimum performance in terms of GFP selectivity (1,237) and yield (98.8%). The optimal system was also successfully scaled up by 50 times without compromising the purification performance. The bottom phase containing GFP was subjected to rotary evaporation of DIMCARB. The stability of GFP was not affected by the distillation of DIMCARB, and the DIMCARB was successfully recycled in three successive rounds of GFP purification. The potential of PPG + DIMCARB + water system as a sustainable protein purification tool is promising.

Economic analysis of the production and recovery of green fluorescent protein using ATPS-based bioprocesses

“…Recent studies were successful in the application of alternative methods such as aqueous twophase systems (ATPS) for the recovery of high purity GFP [28][29][30][31][32][33] or a combination of ATPS and chromatography [18], which can potentially be applied for more cost-effective large-scale processes.…”

Ionic-liquid-based approaches to improve biopharmaceuticals downstream processing and formulation

“…IL’s outstanding properties make them attractive for catalysis, electrochemistry, separation, and extraction, among others ( Fontanals et al, 2009 ; Kianfar and Mafi, 2020 ). Although less investigated, within the pharmaceutical field, ILs have already shown to be important enhancers of the solubility, activity, permeability, and stability of several synthetic drugs ( Pedro et al, 2020 ) and biopharmaceuticals ( Vijayaraghavan et al, 2010 ; Byrne et al, 2012 ; Foureau et al, 2012 ; Kumar and Venkatesu, 2013 ; Mukesh et al, 2013 ; Kumar and Venkatesu, 2014 ; Mazid et al, 2015 ; Todinova et al, 2016 ; Banerjee et al, 2018 ; Jagannath et al, 2018 ; Pedro et al, 2018 ; Reslan et al, 2018 ; Guncheva et al, 2019 ; Lin et al, 2019 ; Quental et al, 2019 ; Mandal et al, 2020 ; Lin et al, 2021 ; Dhiman et al, 2022 ; Vicente et al, 2022 ), and also in the extraction and purification of these biomolecules ( Shu et al, 2010 ; Qu et al, 2012 ; Yuan et al, 2012 ; Chen et al, 2015 ; Ding et al, 2015 ; Ren et al, 2015 ; Taha et al, 2015 ; Ferreira et al, 2016 ; Mondal et al, 2016 ; Ramalho et al, 2018 ; Santos et al, 2018 ; Song et al, 2018 ; Xu et al, 2018 ; Capela et al, 2019 ; Magri et al, 2019 ; Marchel et al, 2019 ; Quental et al, 2019 ; Vicente et al, 2019 ; Castro et al, 2020 ; Neves et al, 2020 ; Vicente et al, 2022 ; Capela et al, 2023 ).…”

Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography

“…Currently, the various strategies, including chromatographic and non-chromatographic techniques, were developed to purify the recombinant GFP or its variants. Nevertheless, it is believed that the purification of GFP with the chromatographic methods generally involves multiple steps, time consuming, low throughput and of high cost [2]. In contrast, ATPS have been viewed as a potential alternative for protein purification because of its cost effectiveness and the simplicity of operation [5].…”

“…Green fluorescent protein (GFP) was originally derived from jellyfish Aequorea victoria species, which exhibit an intensely natural fluorescence [1]. GFP has been regarded as a valuable tool in the field of biology and biotechnology [2]. Due to its widespread application as a molecular biomarker [3, 4], there is an increase in the demand for GFP with high-purity.…”