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

Abstract

BackgroundThe green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity.ResultsThe recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics.ConclusionsThe strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.

Green Fluorescent Protein: discover, structure, purification, applications, and future prospects

“…The hydrophobic effect is amplified in solutions with a high salt content, while it is diminished in solutions with a low salt concentration. Use ammonium sulphate ((NH4)2SO4 instead of other salts because it is highly soluble in water and won't affect most proteins, it turns out [31].…”

Optimising expression and extraction of recombinant proteins in plants

“…These PTGS inhibitory proteins can be co-expressed in plants to increase expression dramatically. Most often, the p19 PTGS inhibitor from tomato bush stunt virus is used (Kuo et al, 2013;Merlin et al, 2014;Dong et al, 2019;Buyel et al, 2021), but others such as the P24 PTGS inhibitor from grapevine leafrollassociated virus-2 (Mardanova et al, 2017), P0 from Polerovirus, P17 from Aureusvirus, or NSs from Tospovirus are also available, though Peyret et al (2019) recently established that P19 is the most effective of these. Ideally, these should always be used when high expression of a RP is desired, in both stable and transient transformants.…”

“…Recombinant protein purification is considered the costliest stage in plant RP production, with comparable costs to other expression systems ( Kuo et al., 2013 ; Merlin et al., 2014 ; Chen and Davis, 2016 ; 2016; Dong et al., 2019 ). Extraction of plant proteins has been reviewed by Wilken and Nikolov (2012) with some further developments in physical ( Buyel and Fischer, 2015 ) and chemical extraction ( Dong et al., 2019 ). Plant protein purification often relies on chromatography techniques, although non-chromatographic techniques also exist and have shown success in plants.…”

Co-expression With Replicating Vector Overcoming Competitive Effects Derived by a Companion Protease Inhibitor in Plants

“…D90197.1) was synthesized and cloned into pCPMV-EV using NruI and FspI restriction sites to generate pCPMV-rPA construct (Figure 1A). For plant-based expression of the inhibitors, synthetic sequences of the PIs gene (NbPR4, HsTIMP, or SlCYS8) were separately cloned into plasmid pCBNoX P19 (Dong et al, 2019) using NcoI and XbaI restriction sites generating pCB301-PIs constructs (Figure 1B). To obtain the replicating rPA expression vector, the rPA expression cassette was cloned into plasmid pJL TRBO-G, a TMV (Tobacco mosaic virus)-based vector (Lindbo, 2007), by PacI and NotI restriction sites to create the pTMV-rPA construct.…”