Plants are an attractive alternative platform for the production of recombinant protein [19]. It offers numerous potential advantages; including low capital equipment, low energy requirements, easy scale-up, reduced risk of carrying pathogen contamination, and ability to post-translational modifications, etc. [9, 19]. Plant viral vectors are widely used as powerful tools for expressing heterologous proteins in plants with inexpensive production costs [20]. Here, we employed the viral expression vector to transiently express recombinant GFP in N. benthamiana, which is commonly used for producing target proteins by plant viral vectors [21]. Using TMV expression vector (pJL TRBO-G) with the help of RNA silencing suppressor, recombinant GFP in soluble form was expressed at an extremely high level (up to ~60% of TSP) in less than one week in the N. benthamiana leaves. In term of the yield, the plant-produced GFP is comparable with that obtained from E. coli (generally ranges from ~10% to ~50% of total protein). Moreover, it has been estimated that purification and downstream processing of recombinant proteins represents 80–90% of the cost of producing pharmaceuticals [22]. GFP could be produced in both soluble form and insoluble inclusion bodies from E. coli over-expression system, depending on culturing conditions (such as low growth temperatures, co-overexpression of molecular chaperones, etc.) [23, 24]. However, ~10% to ~20% of the recombinant GFP was found in the insoluble cell fraction even at optimal conditions [23]. The recombinant proteins produced in E. coli mostly accumulates in inclusion bodies [23] and thus needs to be renatured before purification. The soluble form of plant-produced GFP avoids this tedious and inefficient process of renaturation. Theoretically, the soluble form of plant-produced GFPthe production of GFP in plant may possess a good benefit for the cost reduction of the final GFP product. In addition, plant expression platform is more eco-friendly than the most of non-plant expression systems (such as bacteria, yeast, insect, and mammalian cell cultures, etc.). Altogether, we propose that the viral amplicon-based transient expression system is more suitable for the production of recombinant GFP.
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 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]. In addition, it is notable that alcohol/salt ATPS method is eco-friendly because ethanol and salt can be recycled. Although the yield of purified GFP could be considered modest in our study, alcohol/salt ATPS offers a considerably easier and faster way for purification of recombinant GFP. Otherwise, certain issues need to be considered while using alcohol/salt ATPS method in order to obtain effective GFP separation. Firstly, the soluble protein should be properly diluted before use, since GFP concentration exceeding 1.5 mg/ml can result in GFP co-precipitation with host proteins during phase separation. Secondly, operations should be performed at room temperature, because cooling can cause crystallization of ammonium sulfate. Lastly, exposure to ethanol can lead to a degree of protein denaturation. Thus, the step involving removing ethanol from the water-ethanol mixtures should be performed immediately.
The purpose of using HIC chromatography is initially to concentrate the sample and to remove the residuals of organic solvents and salts which may remain in the aqueous phase. However, we found that this step can further increase the GFP purity (from 89.4% to 96.6 %). We speculate that some compounds, which may not be stained with coomassie dye, were removed by the HIC process. Moreover, we also observed that a little amount of fluorescent proteins were deposited on the top of the HIC column and they cannot be eluted even by low-salt buffer. Comparing with ATPS purified samples (Fig. 3b and 3c),, a doubtful dimer GFP band was missed in the HIC purified sample (Fig. 3e),, indicating that the HIC process probably also remove the oligomer version of recombinant GFP (soluble aggregates) [2425]. One may argue that a single step of HIC can be used to purify GFP because it has advantage of handling large volume of samples and yielding a good result in terms of purity and yield [26]. Unfortunately, plant extracts and homogenates contain unique compositions, such as pigments, phenolics, and etc. [27]. Those compositions, especially phenolics, present obstacles in the downstream processes since they can interact with the target proteins during purification [27]. Moreover, the hydrophobic plant pigments can strongly bind to HIC media, thereby seriously reducing the useful life-time of the media to a few cycles of operation [28]. Otherwise, re-generation of the HIC media with high-frequency is costly since it requires the use of organic solvents [28]. Therefore, pretreatment is highly needed and a single step of HIC might be unattractive in practice for recovery and purification of recombinant proteins from plant tissues.
In this study, a strategy for producing GFP, which combines advantages of both plant virus-based expression platform and alcohol/salt ATPS, was developed. The high level of recombinant GFP was achieved by plant virus-based overexpression vector and the high yield adds to the economy of the downstream process. Moreover, the complete purification process requires only a few steps, takes only ~4 h (Fig. 5) and recovers 34.1% of the protein, equivalent to a yield of ~1.7 g of GFP per kilogram of N. benthaniana leaves. The purity of final GFP product was determined to be more than 95%, and there were no changes in its spectroscopic characteristics. Although we did not experimentally compare our strategy with E. coli-based commercial strategy, two green technologies are involved in our strategy, thus making it more eco-friendly than what of the previously established methodologies. Accordingly, the developed expression and purification process in this study offer a cost-effective and concise alternative for the production of GFP.