Incorporation of regulatory DNA elements within a viral vector improves recombinant protein expression in plants

Plants have significant potential as recombinant protein expression chassis, as they can produce complex post-translationally modified proteins that are unobtainable using prokaryotic production systems, with almost limitless scalability and substantially reduced costs relative to eukaryotic cell cultures. Transient protein expression reduces the time taken between transformation and recombinant protein extraction and purification, however low protein yields relative to conventional stable expression systems remain a major obstacle. Here, we have assessed the effectiveness of combining several established genetic components, including a promoter, 5’ UTR, 3’ UTR, double terminator, and matrix attachment region, to modify the TMV-based pJL-TRBO expression vector for improved recombinant protein expression in plants. Using enhanced green fluorescent protein (eGFP) as a reporter, we quantified expression using fluorescence imaging in planta together with SDS-PAGE and western blotting and showed that our optimum construct resulted in a ~ 7-fold increase relative to pJL-TRBO-eGFP. This increase was exclusively due to the presence of the additional 5’ UTR. We anticipate that our expression constructs will be a useful tool for high-yield plant recombinant protein production and may serve as a template for further improvements.