Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

Lucas Arzola,Karen A Mcdonald,James M Maclean,Kittipong Rattanaporn,Junxing Chen

doi:10.3390/ijms12084975

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.

Highlights

Epidemics of recent emerging infectious diseases such as avian influenza, or more recently theH1N1 pandemic, demand cost-efficient and scalable manufacturing technologies that can rapidly deliver effective therapeutics into the clinical setting
ELISA analysis was used to quantify the capillary morphogenesis 2 (CMG2)-Fc protein that was transiently expressed within the leaves of the N. benthamiana intact plants (Figure 1)
Transient expression in intact plants was observed to be higher with p1 co‐expression at 3.5 days, resulting in a maximum average production of 0.56 g PBI‐220 per kg of leaf fresh weight (FW) and 1.5% of the total soluble protein (TSP)

Summary

Introduction

H1N1 pandemic, demand cost-efficient and scalable manufacturing technologies that can rapidly deliver effective therapeutics into the clinical setting. The threat of bioterrorism makes evident the need for facilitating the production of therapeutic proteins for the mitigation of potential outbreaks. Current manufacturing technologies for therapeutics, such as mammalian cell culture or chicken eggs, are ill-equipped to supply the demand such an outbreak would create, and are not able to produce therapeutics on a large scale in a short amount of time [1,2]. Using plants as production hosts for recombinant proteins is an attractive option because it offers important features such as cost-effectiveness, scalability, and safety. Plant-based production can replace traditional infrastructure required in the upstream portion of manufacturing facilities [6], and several acres of biomass would be sufficient to generate millions of protein doses over the course of a year [7]. Protein expression in plants is adequate for production of human therapeutics, because plants do not harbor or propagate human viruses or pathogens, while still being able to perform protein post-translational modifications [8]

Objectives

Results

Conclusion