| The constant threat of bioterrorism and the massive outbreaks that have occurred for both veterinary and human infectious diseases in the last decade point to the pressing need for manufacturing technologies that enable responsive, scalable, and cost-effective production of therapeutics. In recent years, transient expression in plants has emerged as a feasible alternative to meet this demand. In this dissertation project, a transient production platform that utilized Agrobacterium infiltration of Nicotiana benthamiana plants was developed to express two different proteins: a therapeutic protein and a subunit vaccine.;The expression of a novel anthrax receptor decoy protein, CMG2-Fc, under the control of the constitutive CaMV 35S promoter was analyzed in both Nicotiana benthamiana intact plants and detached leaves. The co-expression of CMG2-Fc was evaluated with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum protein expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in CMG2-Fc production when compared to controls without gene silencing suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI. The expected molecular weight of CMG2-Fc (47.2 kDa) was confirmed through Western Blot.;A transient production platform was also developed for the expression of the hemagglutinin neuraminidase (HN) protein, a subunit vaccine candidate for poultry against Newcastle Disease Virus (NDV). This project evaluated the effect of the psaDb transcriptional enhancer and of the 2S2, RAmy3D, and native HN signal peptides on the protein expression level in Nicotiana benthamiana plants by generating four different constructs for the codon-optimized HN sequence: HN1, HN2, HN3, and HN4. HN protein from all four constructs was produced with p19 co-expression, and was analyzed under the control of the constitutive CaMV 35S promoter, the viral TRBO vector system, and the CMV viral amplicon system. A maximum expression level of 18 mg/kg FW (0.23% of TSP) was observed for the HN1 construct at 6 DPI using the TRBO expression system and p19 co-expression. The expected molecular weight of the four plant-made HN protein variants (63 kDa) was confirmed through Western Blot.;These experiments suggest that the highest protein expression levels in Nicotiana benthamiana can be achieved at 6 days post-infiltration using intact plants as the production host, gene silencing suppression with p1, p19, p21, or p25, and with genetic constructs that contain the psaDb transcriptional enhancer and the 2S2 signal peptide for endoplasmic reticulum retention. This research will help to better understand the transient production of therapeutic proteins and subunit vaccines in Nicotiana benthamiana plants, and will contribute to the development of plant-based manufacturing platforms for the expression of high-value proteins. |
