title: Expression of bioactive anti-CD20 antibody fragments and induction of ER stress response in Arabidopsis seeds.
abstract: Seed-based expression system is an attractive platform for the production of recombinant proteins in molecular farming. Despite the many advantages of molecular farming, little is known about the effect of the different subcellular accumulation of recombinant proteins on the endoplasmic reticulum (ER) quality control system in host plants. In this study, we analyzed the expression of anti-CD20 antibody fragments in seeds of Arabidopsis thaliana (ecotype Columbia) and corresponding glycosylation mutants, and evaluated the influence of three different signal sequences on the expression levels of scFv-Fc of C2B8. The highest protein accumulation level, with a maximum of 6.12 % total soluble proteins, was observed upon fusing proteins to the signal peptide of Arabidopsis seed storage albumin 2. The ER stress responses in developing seeds at 13 days post-anthesis were also compared across different transgenic lines under normal and heat shock conditions. Based on the gene expression profiles of ER stress transducers, our results suggest that accumulation of antibody fragments in the ER exerts more stress on ER homeostasis. In addition, quantitative PCR results also implicate enhanced activation of ER-associated degradation in transgenic lines. Last but not the least, we also demonstrate the anti-tumor potency of plant-derived proteins by showing the anti-tumor activity of purified scFv-Fc proteins against Daudi cells. Together, our data implies that better understanding of the interaction between exogenous protein production and the cellular quality control system of the host plant is necessary for the development of an optimal expression strategy that will be especially beneficial to commercial protein manufacturing.
authors.affiliation: College of Life Sciences, Jilin University, Changchun, 130021, China.
authors.forename: Dezhong
authors.initials: D
authors.lastname: Wang
authors.forename: Jisheng
authors.initials: J
authors.lastname: Ma
authors.forename: Difei
authors.initials: D
authors.lastname: Sun
authors.forename: Haiyan
authors.initials: H
authors.lastname: Li
authors.forename: Chao
authors.initials: C
authors.lastname: Jiang
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D059865:Endoplasmic Reticulum Stress; D015870:Gene Expression; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000633:toxicity; D000069283:Rituximab / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D012639:Seeds / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
chemical_list: D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; D000069283:Rituximab