Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds.
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.
Van Droogenbroeck
Bart
B
Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium.
Cao
Jingyuan
J
Stadlmann
Johannes
J
Altmann
Friedrich
F
Colanesi
Sarah
S
Hillmer
Stefan
S
Robinson
David G
DG
Van Lerberge
Els
E
Terryn
Nancy
N
Van Montagu
Marc
M
Liang
Mifang
M
Depicker
Ann
A
De Jaeger
Geert
G
D017360:Arabidopsis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D010449:Peptide Mapping; D046549:Plantibodies / Q000235:genetics*; D030821:Plants, Genetically Modified; D014357:Trypsin / Q000737:chemistry
D046549:Plantibodies; D014357:Trypsin