A KDEL‐tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds

Petruccelli, Silvana; Otegui, Marisa S.; Lareu, Fabricio Jorge; Dinh, Olivia Tran; Fitchette, Anne‐Catherine; Circosta, Ariana; Rumbo, Martı́n; Bardor, Muriel; Carcamo, R.; Gomord, Véronique; Beachy, Roger N.

doi:10.1111/j.1467-7652.2006.00200.x

Cited by 144 publications

(144 citation statements)

References 88 publications

(134 reference statements)

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“…Probably, the formation of these compartments does not depend on the amount of recombinant protein produced but on specific properties of the recombinant protein itself (Morandini et al, 2011). In the same study, the 65-kD isoform of Glu decarboxylase and proinsulin were, despite bearing a KDEL tag, both localized in the protein storage vacuoles, a phenomenon reported before (Petruccelli et al, 2006;Van Droogenbroeck et al, 2007;Loos et al, 2011a).…”

supporting

confidence: 49%

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

et al. 2012

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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.

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supporting

confidence: 49%

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

et al. 2012

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“…This construct may contribute to a refined knowledge of the sites of glycan modification within the secretory pathway of plants, which may open a wider spectrum of possibilities with important biotechnological applications (Petruccelli et al 2006).…”

Section: Discussionmentioning

confidence: 99%

Expression of a glycosylated GFP as a bivalent reporter in exocytosis

et al. 2009

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The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an a-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a b-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology.

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“…In addition, posttranslational modification occurs in the ER. The KDEL can act as an autonomous retrieval signal and is introduced at the C-terminus for optimum targeting, with accumulation of interesting genes in transgenic plant glycosylation (Agarwal et al, 2008;Petruccelli et al, 2006). N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural forms (Lerouge, 1998).…”

Section: Discussionmentioning

confidence: 99%

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

Kim¹,

Youm²,

Lee³

et al. 2014

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Transgenic potato was generated to express recombinant 5 repeated β-amyloid (Aβ) peptides, potential antigens to be applied as a preventive accine for Alzheimer's disease using Agrobacterium mediated transformation. The Aβ peptides were fused to the human IgG Fc fragment enhancing protein and KDEL, which is the endoplasmic reticulum (ER) retention signal (5Aβ-FcK). The 5Aβ-FcK, was expressed under the control of the duplicated 35S promoter. PCR analysis confirmed the presence of the transgene in several transgenic potato lines. Southern blot analysis showed only a single gene copy number in transgenic line 22, whereas multiple gene copy numbers were shown for transgenic lines 31 and 44. Northern blot analysis showed that line 22 had stronger mRNA levels when compared to lines 31 and 44. Immunoblot analysis confirmed that the 5Aβ-FcK protein was expressed in the transgenic potato plant. These results indicate that 5Aβ fused to Fc can be expressed in potato plants.

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A KDEL‐tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds

Cited by 144 publications

References 88 publications

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

Expression of a glycosylated GFP as a bivalent reporter in exocytosis

Expression of Aβ-Fc Fusion Protein in Transgenic Potato

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