title: 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.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of California, Davis, One Shields Ave, Davis, CA 95616, USA; E-Mails:  larzola@ucdavis.edu  (L.A.);  cicchen@ucdavis.edu  (J.C.);  krattanaporn@ucdavis.edu  (K.R.).
authors.forename: Lucas
authors.initials: L
authors.lastname: Arzola
authors.forename: Junxing
authors.initials: J
authors.lastname: Chen
authors.forename: Kittipong
authors.initials: K
authors.lastname: Rattanaporn
authors.forename: James M
authors.initials: JM
authors.lastname: Maclean
authors.forename: Karen A
authors.initials: KA
authors.lastname: McDonald
mesh_terms: D015870:Gene Expression; D020868:Gene Silencing; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism
chemical_list: C447150:ANTXR2 protein, human; D007141:Immunoglobulin Fc Fragments; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C070309:anthrax toxin receptors; D014841:von Willebrand Factor