Protein Targeting and Vesicular Trafficking
Ahmad, Adil , Menassa, Rima .
Expression of Therapeutic Proteins in Tobacco BY-2 Cell Suspension Culture.
In recent studies, many recombinant proteins have been expressed in plants for the production of therapeutic pharmaceuticals including cytokines, antibodies, recombinant enzymes and human vaccines. The levels of accumulation of these proteins in their respective plant systems are variable. The human interleukin-10 protein (IL-10) is an 18.5 kDa homodimeric protein that possesses anti-inflammatory properties and is a potential treatment for inflammatory bowel disease. IL-10 is a good representative of other therapeutic pharmaceuticals, but does not accumulate to a desirable level in tobacco leaves. We have previously shown that IL-10 targeted to the ER accumulated to levels of 50 ng/mg of total soluble protein (TSP). The same construct produced 250 ng of IL-10 per mg of TSP when expressed in tobacco BY-2 cells. The use of transgenic BY-2 cell lines for the stable in vivo production of secreted IL-10 thus provides a viable model for the expression and suitable accumulation of plant-made pharmaceutical proteins. In an effort to simplify purification and reduce its cost, we created constructs that would secrete IL-10 to the BY-2 culture medium. Preliminary results showed levels of IL-10 fused to an ELP tag in transgenic BY-2 calli ranging from 0.18 to 109 ng/mg TSP. High expressing calli will be used to produce stable, perpetual cell lines for the examination of accumulation levels, sub-cellular localization, the rate of protein turnover, and optimization of growth conditions of BY-2 cell lines for the economical purification of this therapeutic protein. The recovery of recombinant IL-10 may be further enhanced by modifying the culture medium through the addition of various agents including gelatin, bovine serum albumin and polyvinylpyrrolidone. These agents are thought to protect the target protein from degradation, prevent precipitation and minimize adsorption to vessel walls, thus increasing product yield by addition of low cost stabilizers.
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1 - University of Western Ontario, Department of Biology, Biological & Geological Sciences Building, University of Western Ontario, London, Ontario, N6A 5B7, Canada
2 - Agriculture and Agri-Food Canada, 1391 Sandford St., London, Ontario, N5V 4T3, Bioproducts and Bioprocesses
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM