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Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds

机译:转基因拟南芥种子中高度积累的单链Fv-Fc抗体的异常定位和糖基化

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摘要

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 ribosome-associated 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 protein-sorting mechanisms in the secretory pathway.
机译:在转基因种子中生产高价值的重组蛋白是基于哺乳动物细胞和细菌的常规系统的一种有吸引力且经济上可行的替代方案。与叶相反,种子允许重组蛋白在相对较小的体积和稳定的环境中大量积累。我们证明了具有N端信号序列和C端KDEL标签的单链可变片段(scFv)-Fc抗体可以作为拟南芥种子中的二价IgG样抗体积聚到非常高的水平,并说明了植物产生的抗甲型肝炎病毒scFv-Fc与相应的全长IgG具有相似的抗原结合和体外中和活性。如所预期的,种子中产生的大多数scFv-Fc仅包含寡甘露糖型N-聚糖,但是出乎意料的是,35-40%从未糖基化。在由核糖体相关膜界定的内质网(ER)的隔室中发现了一部分scFv-Fc。此外,与糖基化数据一致,大量重组蛋白沉积在周质空间中,这意味着从ER直接转运到质膜和细胞壁之间的周质空间。 ER伴侣钙网蛋白和结合蛋白(BiP)和内源种子存储蛋白cruciferin在周质空间中的异常定位表明重组scFv-Fc的过量生产会干扰正常的ER保留和分泌途径中的蛋白分选机制。

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