Expression of recombinant Green Fluorescent Protein (GFP) using ‘magnICON®’ system in-planta

The green fluorescent protein (GFP) is widely used as a reporter for gene expression and localisation, as an in situ tag for fusion proteins, a biosensor or a probe for protein-protein interactions. It is 238 amino acids long (26.9 kDa) and forms a ß-barrel around one α-helix. GFP isolated from the jelly fish Aequorea victoria and demonstrated that it could exhibit bright green fluorescence when exposed to light in the UltraViolet (UV) range. This study focused on the rapid transient expression of GFP using the deconstructed tobacco mosaic virus-based ‘magnICON®’ plant expression system. A 3’ viral vector containing the GFP gene (pICH7410) was infiltrated into N. benthamiana leaves side by side with the 5’ module for cytosol or the 5’ module for chloroplast. At 10 days post infiltration (dpi), GFP expression in leaves was checked under UV light to ensure the viral vector-based heterologous expression was working correctly. GFP protein showed bright fluorescence under UV light. The TSP extracted from infiltrated leaves using 3’ GFP module and different 5’ modules (cytosol or chloroplast) were quantified using Bradford assay. The absorbance of each sample was read at 595 nm and the TSP concentration determined from the standard curve, constructed using bovine serum albumin (BSA). Approximately 93 μg and 98 μg of the quantified TSP samples were run on a gel to analyse the GFP expression by SDS-PAGE gel and western blot. Western blot analysis using anti-GFP antibody confirmed the presence of GFP at approximately 25 kDa, at the expected size for GFP.

کلیدواژه ها:

green fluorescent protein (GFP) ، transient expression ، magnICON