Expression of a functional antibody in Nicotiana tabacum plants and culture cells : analysis of proteolytic products and identification of putative extracellular peptidases involved
(2009)
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- Abstract
- Plants represent an interesting alternative host for expressing pharmacological proteins. However, like other expression systems, plants produce peptidases that might degrade ectopic proteins. We have expressed LO-BM2, a therapeutic IgG antibody, in N. tabacum plants and BY-2 suspension cells. Two different expression constructs were tested in which genes encoding the antibody heavy and light chains were assembled in the tandem or inverted convergent orientation. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Antibody purified from leaf tissues and BY-2 cells was functional. However, mass spectrometry analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted out of the BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment. We then performed the identification of N. tabacum peptidases possibly responsible for antibody degradation within the extracellular medium of BY-2 cells. By IgG zymography and the use of peptidase inhibitors, we showed that a serine peptidase was capable of degrading antibodies. By two-dimension electrophoresis and mass spectrometry analysis, this peptidase was shown to be a close homolog of Solanum lycopersicum SlSBT4E subtilisin-like peptidase. Three full-length and one partial cDNA encoding N. tabacum subtilisin-like peptidases were cloned and showed to share high identity with the members of the S. lycopersicum peptidase SlSBT3/SBT4 subfamily.
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De Muynck, B. (2009). Expression of a functional antibody in Nicotiana tabacum plants and culture cells : analysis of proteolytic products and identification of putative extracellular peptidases involved. https://hdl.handle.net/2078.5/124045