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

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]Clbased 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 (backextraction) 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.

Economic evaluation of M13 bacteriophage production at large‐Scale for therapeutic applications using aqueous Two‐Phase systems

“…22 This is due to the essence of their typical construction, where the sample being separated usually represents a 10 to 30% w/w (sample loading). This means that system components constitute approximately 70 to 90% w/w of a system, as this has been reported before 27,28,30 and employed in this study. In an industrial scale, bioprocesses where a 1000 L bioreactor is operated, if the culture media is used directly after fermentation into ATPS as sample (Fig.…”

“…As mentioned before, current research focuses on using low sample loading 27,28 and this parameter is not frequently optimized, but from an economic perspective, it might be relevant to study its effect. Results presented here show that recovery yield decreases as sample loading increases, but this effect can be product-or sample-related.…”

Revealing a new fluorescence peak of the enhanced green fluorescent protein using three-dimensional fluorescence spectroscopy

“…Enhanced Green Fluorescent Protein (EGFP) with $97% purity was produced, puried and quantied using methods previously developed by the group and detailed in dos Santos et al 2018, using Escherichia coli BL21 (DE3) with plasmids pLysS and pET28(a), aqueous biphasic system and ultraltration. 17 The equivalency of the EGFP standard from the Biovision® quanti-cation kit and the EGFP produced and puried in this study was conrmed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), detailed in the ESI, Section S.1. †…”

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

“…Through the application of DNA recombinant technology, GFP has successfully been produced by a variety of hosts [5]. Currently, the commercially available GFP produced by Escherichia coli costs approximately US$ 2,000.00 per mg [6]. A cost-efficient upstream expression system and an inexpensive downstream purification process will be able to reduce the production costs and thereby meet the demands of the GFP with high-purity.…”