Protein Targeting and Vesicular Trafficking
Kaldis, Angelo , Menassa, Rima .
Trafficking and degradation of recombinant IL-10 in plants.
During the past few years, a wide variety of recombinant proteins have been expressed in plants. Some proteins such as the reporter proteins GUS and GFP accumulate to high levels, while others accumulate to very low levels in plants. Inherent properties of these proteins, protein turnover rates, as well as their sub-cellular localization affect their accumulation levels. The human interleukin-10 protein (IL-10) is a labile protein with a half-life in vivo of 30 minutes. It requires post-translational modifications and assembly, and is a good representative of other therapeutic proteins. However, IL-10 does not accumulate in plants to levels desirable for therapeutics production. To develop an understanding of the regulation of IL-10 fate in plant cells, we used tobacco BY-2 cells stably transformed with the same ER-targeted construct that we had used in transgenic plants. We found that BY-2 cells produce about 5 times more IL-10 per mg of TSP than transgenic plants, and after subjecting transformed BY-2 cells to different inhibitor treatments, it appeared that IL-10 is degraded by proteases, but not the proteasome. To define the sub-cellular compartment of IL-10 degradation, we produced an ER-targeted fusion of IL-10 with GFP, and observed the localization of the fusion protein by confocal microscopy. The fusion protein was observed in a reticulated pattern typical of the ER after 3 days of growth. However, GFP fluorescence intensified as the cells progressed through the 7-day cycle, peaking and appearing in the vacuole on day 6. Conversely, quantitation by ELISA revealed that IL-10 levels reached a maximum 3 days after sub-culturing, followed by a steady decline in subsequent days. Further examination of the fusion protein by immunoblotting revealed cleavage between IL-10 and GFP, allowing accumulation of GFP while the IL-10::GFP fusion levels declined steadily and no IL-10 protein on its own was detectable.
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1 - Agriculture and AgriFood Canada, Bioproducts and Bioprocesses, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada
2 - Agriculture and Agri-Food Canada, Bioproducts and Bioprocesses, 1391 Sandford Street, London, Ontario, N5V 4T3, Canada
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM