Research Article

Plant Molecular Biology

, Volume 30, Issue 4, pp 781-793

First online:

The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco

  • Alexander SchoutenAffiliated withDepartment of Nematology, Wageningen Agricultural University
  • , Jan RoosienAffiliated withLaboratory for Monoclonal Antibodies
  • , Fred A. van EngelenAffiliated withDepartment of Molecular Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLO)
  • , G. A. M. (Ineke) de JongAffiliated withDepartment of Nematology, Wageningen Agricultural University
  • , A. W. M. (Tanja) Borst-VrenssenAffiliated withDepartment of Nematology, Wageningen Agricultural University
  • , Jacoline F. ZilverentantAffiliated withLaboratory for Monoclonal Antibodies
  • , Dirk BoschAffiliated withDepartment of Molecular Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLO)
  • , Willem J. StiekemaAffiliated withDepartment of Molecular Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLO)
  • , Fred J. GommersAffiliated withDepartment of Nematology, Wageningen Agricultural University
    • , Arjen SchotsAffiliated withLaboratory for Monoclonal Antibodies
    • , Jaap BakkerAffiliated withDepartment of Nematology, Wageningen Agricultural University

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Abstract

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.

Key words

Genetic engineering KDEL retention signal signal peptide single-chain antibody targeting