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.

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

“…The expression of hGAD65mut in transgenic tobacco plants has been previously reported 12 , showing that recombinant protein levels varied significantly among independently transformed lines. The best-performing hGAD65mut T1 transgenic plant was self-crossed and the derived plants (T2) were analyzed to select again the best performing one.…”

High-level transient expression of the N-terminal domain of IpaD from Shigella dysenteriae in four plant species transformed with different construct configurations

“…It has been also mentioned that PTGS plays a key role in shortening the transient expression period (Voinnet et al 2003). Meanwhile, according to the studies of Arzola et al (2011) andVoinnet et al (2003), the coinfection of infiltrated leaves with a viral-silencing suppressor may improve the transgene expression level. It has been also reported that the use of 2b derived from CMV could enhance expression levels of different proteins in N. benthamiana up to 5.5-fold (Moon et al 2012).…”

“…In addition, two signal peptides, Zera ® (Mainieri et al 2014) andExtensin (De Loose et al 1991), were utilized to examine the potential roles of each on the accumulation of the recombinant IpaD protein in the plants under study. Meanwhile, an approach based on suppression of posttranscriptional gene silencing (PTGS) was employed using Cucumber mosaic virus (CMV) 2b protein (Goto et al 2007;Arzola et al 2011) to determine its effect on the production quantity of the current recombinant IpaD protein.…”

From Pandemic Preparedness to Biofuel Production: Tobacco Finds Its Biotechnology Niche in North America

“…Lastly, the DNA plasmid constructs engineered by researchers to be transferred from bacteria into plants have historically incorporated or built upon genetic elements of tobacco mosaic virus (TMV) or other plant viruses that replicate in tobacco leaves, therefore further justifying the use of tobacco as a plant host for expression of these target biotherapeutics. [Mallajosyula, 2014: 24378714], [Kalthoff, 2010: 20810729], [Kanagarajan, 2012: 22442675] Ebola virus [12][13][14][15] [ Phoolcharoen, 2011: 21281425], [Phoolcharoen, 2011: 22143779], [Huang, 2010, 20047189], [Castilho, 2011: 22039433] Dengue [16][17][18][19][20] [ Kim, 2015: 25728317], [Conconi-Linares, 2013: 23499580], [Saejung, 2007: 17659815], [Martinez, 2010: 20213522], [Martinez, 2012: 22480936] Norovirus [21][22][23][24][25] [ Mathew, 2014: 24949472], [Santi, 2008: 18325641], [Lai, 2012: 22134876 [Farrance, 2011: 21715576], [Feller, 2013: 24278216], [Kapelski, 2015: 25651860], [Voepel, 2014: 25200253], [Clemente, 2012: 22911156], [Beiss, 2015: 25615702], West Nile Virus [49][50][51] [ Chen, 2015: 25676782], [Lai, 2014: 24975464], 24675995] Bacillus anthracis (anthrax) [52][53][54][55][...…”