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.
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.
Schouten
A
A
Wageningen Agricultural University, Department of Nematology, The Netherlands.
Roosien
J
J
van Engelen
F A
FA
de Jong
G A
GA
Borst-Vrenssen
A W
AW
Zilverentant
J F
JF
Bosch
D
D
Stiekema
W J
WJ
Gommers
F J
FJ
Schots
A
A
Bakker
J
J
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002265:Carboxylic Ester Hydrolases / Q000276:immunology; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010947:Plants, Toxic; D015202:Protein Engineering / Q000379:methods; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; C073762:lysyl-aspartyl-glutamyl-leucine; D002265:Carboxylic Ester Hydrolases; C019952:cutinase