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

Cher Pin Song,Chien Wei Ooi,Poh En Liew,Schian Pei Lim,Zora Teh,Pau Loke Show

doi:10.3389/fchem.2018.00529

Cher Pin Song, Chien Wei Ooi + Show 4 more

Open Access

https://doi.org/10.3389/fchem.2018.00529

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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.

Highlights

Green fluorescent protein (GFP) has been widely applied in the cellular and molecular biology research due to its unique properties such as the intense fluorescence visibility, high thermal stability, and the adjustable fluorescence intensity via a proper manipulation of the protein structure (Skosyrev et al, 2003; Li et al, 2009; Quental et al, 2015)
The PPG + DIMCARB + water systems were successfully applied for the purification of green fluorescent protein (GFP) from the clarified E. coli lysate
GFP has a higher affinity toward the DIMCARBrich phase

Summary

Introduction

Green fluorescent protein (GFP) has been widely applied in the cellular and molecular biology research due to its unique properties such as the intense fluorescence visibility, high thermal stability, and the adjustable fluorescence intensity via a proper manipulation of the protein structure (Skosyrev et al, 2003; Li et al, 2009; Quental et al, 2015). The advantages of ATPS include the high extraction efficiency, the cost effectiveness, and the simplicity of operation. This type of liquid-liquid extraction is commonly exploited for the primary recovery and purification of valuable biological products such as proteins (Merchuk et al, 1998), enzymes (Kroner et al, 1982), nucleic acids (Gomes et al, 2009), and viruses (Liu et al, 1998). The extraction of GFP has been successful achieved using the traditional ATPSs consisting of phase-forming components such as polymer, surfactant, alcohol, and inorganic salts (Jain et al, 2004; Johansson et al, 2008; Li and Beitle Robert, 2008; Samarkina et al, 2009; Lopes et al, 2011; Lo et al, 2018). The limited polarity range of the coexisting phases and the poor recyclability of the conventional phase-forming components have constituted a major bottleneck that hampers the vast use of these conventional ATPSs (Hatti-Kaul, 2000)

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