Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein

Abstract

Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc 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 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 expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI.

Keywords: Nicotiana benthamiana; anthrax; fusion protein; gene silencing suppressors; molecular farming; transient.

Figure 1.

ELISA analysis of transiently expressed CMG2-Fc on a leaf FW basis in intact plants in which the p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro PTGS suppressors were co-expressed. An infiltration of CMG2-Fc without PTGS suppressor co-expression was also performed to have a baseline for comparison. CMG2-Fc expression levels are the average of triplicate N. benthamiana leaves exposed to the same condition. Error bars are based on the propagation of errors calculated from triplicate assays performed on samples of triplicate N. benthamiana leaves from the intact plants.

Figure 2.

Western Blot analysis of CMG2-Fc protein from intact N. benthamiana plants reduced with 90 mM dithiothreitol (DTT). Samples shown below were obtained from plants harvested at either 3.5 or 14 days post-infiltration (DPI). Lanes 1 and 2 show CMG2-Fc expression on intact plants: CMG2-Fc co-expressed with gene silencing suppressor p1 at 3.5 DPI (lane 1); CMG2-Fc without gene silencing suppressor co-expression at 3.5 DPI (lane 2). Lanes 3 and 4 show CMG2-Fc expression on detached leaves: CMG2-Fc co-expressed with gene silencing suppressor p19 at 14 DPI (lane 3); CMG2-Fc without gene silencing suppressor co-expression at 14 DPI (lane 4). Lane M: molecular weight marker; lane WTi: control infiltration of an intact plant (without Agrobacterium); lane WTd: control infiltration of a detached leaf (without Agrobacterium); lane S: purified CMG2-Fc protein standard (2000 ng). Purified CMG2-Fc was used as a positive control to allow comparisons between the relative band intensities. Approximate sizes (kDa) of proteins are shown.

Figure 3.

ELISA analysis of transiently expressed CMG2-Fc on a leaf FW basis in detached leaves co-expressed with p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro PTGS suppressors. An infiltration of CMG2-Fc without PTGS suppressor co-expression was performed to have a baseline for comparison. CMG2-Fc expression levels are the average of triplicate N. benthamiana leaves exposed to the same condition. Error bars are based on the propagation of errors calculated from triplicate assays performed on samples of triplicate N. benthamiana leaves.