Expression Of Bioactive Antibody Fragments And Induction Of ER Stress Response In Arabidopsis Seeds

Molecular farming is the production of recombinant pharmaceuticals, industrialproteins and secondary metabolites in plants. Nowadays, there are a lot of mammalianderived proteins that are expressed in plants including human serum albumin,hormones, monoclonal antibody, vaccine and cytokines.Among the different platforms of molecular farming, seed-based expressionsystem is an attractive platform for the production of recombinant proteins. As naturalstorage organs, seeds provide stable environment for recombinant proteinaccumulation, and convenient in transport. Despite the many advantages of molecularfarming in recent years, little is known about the effect of the different subcellularaccumulation of recombinant proteins on the endoplasmic reticulum quality controlsystem in host plants.In this study, we analyzed the expression of anti-CD20antibody fragments inseeds of Arabidopsis thaliana (ecotype Columbia-0) and corresponding glycosylationmutants (TKO). Firstly, signal sequences of barley α-amylase,2S2albumin ofArabidopsis thaliana and coding sequence of SEKDEL were added to the codingsequence of scFv-Fc by fusion PCR and four plant expression vectors with differentregulatory motif were obtained.181transgenic individuals were identified afterselection on phosphinothricin containing Murashige and Skoog agar medium. And weevaluated the influence of three different signal sequences on the expression levels ofscFv-Fc of C2B8by Elisa, SDS-PAGE and Western Blot. The highest proteinaccumulation level, with a maximum of6.12%total soluble proteins, was observedupon fusing proteins to the signal peptide of Arabidopsis seed storage albumin2.Furthermore, the N-glycan structures of recombinant scFv-Fc were analyzed bydigestion with Endo H and PNGase F. scFv-Fc fromWTC2B8SEKDELcarried highmannose type N-glycans, and the N-glycoform of antibody fragments from plants of TKO background lack fucose modification and that parts of the scFv-Fc containedhigh mannose residues. The scFv-Fcs from wild type seeds contained the complexN-glycan modification with α1,3-fucose We also demonstrate the anti-tumor potencyof plant-derived proteins by showing the anti-tumor activity of purified scFv-Fcproteins against Daudi cells. Enhanced ADCC activity was observed when scFv-Fcproteins from plants of TKO background were used, and this observation is due toafucosylated antibodies enhance the binding activity of FcγRIIIa receptor. The CDCresults demonstrated that plant-derived scFv-Fcs, although slightly less potent thanRituximab, could kill tumor cells efficiently via the CDC pathwayThe ER stress responses in developing seeds at13days post anthesis were alsocompared across different transgenic lines under normal and heat shock conditions.Based on the gene expression profiles of ER stress transducers, our results suggestthat accumulation of antibody fragments in the ER exerts more stress on ERhomeostasis. In addition, quantitative PCR results also implicate enhanced activationof ER-associated degradation in transgenic lines. Due to the potential disturbance ofcellular homeostasis triggered by the expression of recombinant protein could affectprotein synthesis, modification and secretion, better understanding of the interactionbetween exogenous protein production and the cellular QC system of the host plant isnecessary for the development of an optimal expression strategy that will beespecially beneficial to commercial protein manufacturing.