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

Abstract

Background: The 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. Results : The recombinant GFP was transiently expressed in an active form in agoinoculated N. 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. Conclusions: The 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. Keywords: Green fluorescent protein, Plant virus, Transient gene expression, Aqueous two-phase system, Hydrophobic interaction chromatography

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

“…Envisaging the future implementation of ATPS 7-based bioprocess in the purification of GFP, if a high level of purity is needed for whichever application, additional chromatographic operation units will need to be incorporated. In literature, some studies have successfully applied chromatographic operations to purify GFP [18,27], while others have already addressed the cost implication of incorporating chromatographic operations [53] for purification of proteins. Although there is no economic evaluation of chromatography processes for purifying GFP, the economic evaluation of a bioprocess for production of uricase clearly demonstrated that the inclusion of chromatography generates a large build up in the costs associated to the capital investment and consumables, as more complex equipment is needed and resins are more expensive than phase-forming chemicals [53].…”

“…In the last years, some studies are aiming to decrease GFP price by developing new ways to produce it successfully [15,16]. Mainly two strategies are in place, with the first one focusing on improvements in upstream processes by obtaining high titres using recombinant Escherichia coli, the current available commercial product, [17] or tobacco plants [18] to express the protein. Alternatively, there are also studies aiming to reduce costs by improving the recovery and purification techniques in the downstream process as they can represent a large fraction of the production costs [19,20].…”

“…Therefore, the purification of pharmaceutical biocompounds is usually complex and based in multi-step platforms [22]. For example, the current strategies for GFP purification involve, mainly, the use of multiple chromatographic units, which are complex and costly [18,[23][24][25][26][27].…”

“…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.…”