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.
Highlights
The green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics
A protein corresponding to the expected molecular weight (27 kDa) was detected in the total soluble proteins extracted from the inoculated leaf tissues by both Coomassie stained polyacrylamide gel (Fig. 2c) and Western blot analysis (Fig. 2d)
All results together indicated that the recombinant GFP was successfully and efficiently expressed in the N. benthamiana leaves
Summary
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. Due to its widespread application as a molecular biomarker [3, 4], there is an increase in the demand for GFP with high-purity. 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. It was estimated that proteins can be produced in plants at a cost of 10–50 fold less than in Escherichia coli [8]. Virus-based expression system can express the target proteins in plants at an extremely high level because of viral
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