High-Level Production of a Recombinant Protein in Nicotiana benthamiana Leaves Through Transient Expression Using a Double Terminator

Various bio-based recombinant proteins have been produced for industrial, medical, and research purposes. Plants are potential platforms for recombinant protein production because of several advantages. Therefore, establishing a system with high target gene expression to compensate for the low protein yield of plant systems is crucial. In particular, selecting and combining strong terminators is essential because the expression of target genes can be substantially enhanced. Here, we aimed to quantify the enhancement in the fluorescence intensity of the turbo green fluorescence protein (tGFP) caused by the best double-terminator combinations compared to that of the control vector using agroinfiltration in <i>Nicotiana benthamiana</i> leaves. tGFP fluorescence increased by 4.1-fold in leaf samples infiltrated with a vector containing a double terminator and markedly increased by a maximum of 23.7-fold when co-infiltrated with the geminiviral vector and P19 compared to that in constructs containing an octopine synthase terminator. Polyadenylation site analysis in leaf tissues expressing single or dual terminators showed that the first terminator influenced the polyadenylation site determination of the second terminator, resulting in different polyadenylation sites compared with when the terminator is located first. The combination of the high-expression terminators and geminiviral vectors can increase the production of target proteins.