Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenicArabidopsisseeds

Abstract

Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosomeassociated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and proteinsorting mechanisms in the secretory pathway.glycosylation ͉ molecular farming ͉ recombinant antibody ͉ subcellular localization T ransgenic plants for the production of high-value recombinant proteins are a promising alternative to conventional recombinant protein production systems, such as bacteria, yeast, animal, and insect cell cultures (1). One of the most important factors driving research in this field is yield improvement, because of its significant impact on economic feasibility (2). Strategies to increase recombinant protein yield in plants include development of better expression cassettes, improvement of protein stability and accumulation by using specific subcellular targeting signals, and development of downstream processing technologies (3). In this perspective, seed-based platforms are particularly attractive because they allow recombinant proteins to stably accumulate at a relatively high concentration in a compact biomass, which is beneficial for extraction and downstream processing (4). By using a seed-specific expression cassette based on the regulatory signals of seed storage proteins of common bean (Phaseolus vulgaris), and by targeting the recombinant protein to the endoplasmic reticulum (ER), we obtained the highest yields of recombinant proteins in plants described so far: a single-chain variable fragment (scFv) accumulated to levels in excess of 36% of total soluble protein (TSP) in homozygous Arabidopsis seeds, while retaining its antigen-binding activity and affinity (5).For some applications, fusion of the scFv with the Fc doma...

SYP71, a Plant-specific Qc-SNARE Protein, Reveals Dual Localization to the Plasma Membrane and the Endoplasmic Reticulum in Arabidopsis

“…This membrane-traffic process is mediated by an ER-localized SNARE protein called syntaxin 18 (Hatsuzawa et al, 2000). In transgenic Arabidopsis seed cells, the recombinant single-chain Fv-Fc antibodies are transported directly from the ER to the periplasmic space (Van Droogenbroeck et al, 2007). The SYP71 might be involved in a similar direct pathway from the ER to the PM at least in certain condition/type of plant cells, such as rapidly elongating or dividing cells.…”

Cost-effective production of a vaginal protein microbicide to prevent HIV transmission

“…The MS-data from the tryptic peptides were analyzed as described in ref. 49 and compared with datasets generated by in silico tryptic digestion of the 2G12 coding, using the PeptideMass program (available at www.expasy.org/ tools/peptide-mass.html). Based on the tryptic peptide datasets, tryptic glycopeptide datasets were generated by the addition of glycan mass increments to the masses of the two identified glycopeptides.…”

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response

“…For instance, De Jaeger and coworkers (2002) demonstrated that an expression cassette using the regulatory sequences of ARCELIN5-I and b-PHASEOLIN of bean (Phaseolus vulgaris) could boost the accumulation of a single-chain Fv (scFv) up to 36.5% of the total soluble protein (TSP). Using the same cassette, a single-chain antibody fragment fused to an Fc fragment (scFv-Fc) resulted in the production of 10% bivalent functional antibodies of the TSP in Arabidopsis (Arabidopsis thaliana) seeds (Van Droogenbroeck et al, 2007). Immunolocalization studies on one of these scFv-Fcexpressing lines revealed an aberrant localization of the recombinant scFv-Fc, as well as of endoplasmic reticulum (ER) chaperone binding proteins (BiPs) and calreticulin, and of the endogenous SSP cruciferin, suggesting that the cellular homeostasis was disturbed.…”