Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response    

Abstract

Among the many plant-based production systems that are being tested for molecular farming, seeds are very attractive, as they provide a stable environment in which the accumulating recombinant proteins can be stored. However, it is not known exactly how high production levels of recombinant antibodies influence the endogenous transcriptome and proteome of the developing seed. To address this question, we studied the transcriptomic status in developing Arabidopsis (Arabidopsis thaliana) seeds 13 d post anthesis of three transgenic lines, producing varying levels of recombinant VHH or single-chain Fv antibody fragments fused to the human immunoglobulin G1-derived Fc fragment under the control of the b-PHASEOLIN seed-specific promoter. Using genome-wide Tiling arrays, we demonstrated that only a small proportion of the transcriptome was significantly changed in each of the lines compared with the wild type. Strikingly, in all three lines, we found a large overlap of up-regulated genes corresponding to protein folding, glycosylation/modification, translocation, vesicle transport, and protein degradation, suggestive of a state of cellular stress called the unfolded protein response. Moreover, the gene up-regulation amplitude was similar in all three lines. We hypothesize that the production of recombinant antibodies in the endoplasmic reticulum triggers endoplasmic reticulum stress, causing a disturbance of the normal cellular homeostasis.

Efficient recovery of recombinant proteins from cereal endosperm is affected by interaction with endogenous storage proteins

“…S1), suggesting the onset of ER stress and, indirectly, the activation of the UPR cascade. The UPR cascade was triggered in Arabidopsis thaliana seeds expressing a recombinant scFv-Fc antibody, reflecting the disruption of ER homeostasis [47]. Furthermore, the interaction between free antibody chains and endogenous storage proteins could also induce the expression of chaperones, as previously reported in rice seeds expressing a recombinant version of human interleukin 10 (IL-10) that also interacted with cysteine-rich prolamins [38].…”

Role of plant expression systems in antibody production for passive immunization

“…It is reasoned that the seed-specific promoters are usually active during the seed filling stage (development and maturity), while the CaMV P35S ceases expression after the initial stages, explaining the lower accumulation (Chen et al, 1986, Petruccelli et al, 2006. Besides the b-phaseolin promoter (De Jaeger et al, 2002, De Wilde et al, 2013, several other seed-specific promoters, like USP (unknown seed promoter) (Zimmermann et al, 2009), glutelin-1 (gt-1) promoter (Ramessar et al, 2008), legumin A promoter , and maize ubiquitin promoter (Christensen and Quail, 1996), have been evaluated for the expression of antibodies and other therapeutic proteins in plants ( Khan et al, 2012).…”

“…Although the use of the constitutive Cauliflower mosaic virus (CaMV) 35S promoter (P35S) to drive transgene expression can yield high amounts of antibodies in various plant tissues, its strength in controlling expression in seeds is poor, and a seedspecific promoter, like the b-phaseolin promoter leading to high accumulation is required (De Jaeger et al, 2002, De Wilde et al, 2013, Van Droogenbroeck et al, 2007. It is reasoned that the seed-specific promoters are usually active during the seed filling stage (development and maturity), while the CaMV P35S ceases expression after the initial stages, explaining the lower accumulation (Chen et al, 1986, Petruccelli et al, 2006.…”

“…Alternatively, for parenteral application, the high protein concentration in desiccated seeds facilitates the downstream processing. Different full-length antibody and antibody formats, like ScFv, ScFv-Fc, VHHs and VHH-Fc, have been expressed effectively in seeds of both monocot and dicot plants (De Wilde et al, 2013, Khan et al, 2012, Loos et al, 2011b, Van Droogenbroeck et al, 2009, Virdi et al, 2013.…”

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into theN-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

“…The classical protein secretory pathway is less characterized in plants than in mammals or yeast but is of growing interest due to the potential of plant systems as hosts for the production of recombinant proteins (Erlendsson et al, 2010;De Wilde et al, 2013). Plant secretomics, therefore, is a rapidly expanding area applied to gain further insight into these processes (Agrawal et al, 2010;Alexandersson et al, 2013).…”