Date of Award
2008
Degree Type
Thesis
Degree Name
Master of Science
Program
Biology
Supervisor
Dr. Rima Menassa
Second Advisor
Dr. Susanne Kohalmi
Third Advisor
Dr. Mark Bernards
Abstract
The accumulating body of research over the past two decades suggests that plant cell culture is an economically viable system for the production of protein based drugs. In an effort to investigate alternate routes of protein accumulation, interleukin-10 directed by its native signal peptide was fused to various tags including green fluorescent protein, Strepll and elastin-like polypeptide for the purpose of secretion into a culture medium. Our data suggest that media supplemented with several protein stabilizing agents enhanced the accumulation of secreted interleukin-10. It was also noted that the passage of IL-10 fusions through the cell wall into the extracellular media is limited to a cutoff of ~30 kDa. To elucidate the effect of ELP fusions on IL-10 trafficking and turnover, subcellular localization was performed via confocal analysis. IL-10- GFP-ELP targeted for secretion was found in the vacuoles rather than the apoplast, whereas an endoplasmic reticulum retained version of this construct resulted in the formation of numerous fluorescent protein bodies. The latter construct accumulated levels of IL-10 upwards of 2.4% TSP, 500-fold higher than the secreted identical protein. These results suggest that endoplasmic reticulum-retention is a more viable alternative for the production of plant recombinant IL-10 when compared to secretion.
Recommended Citation
Ahmad, Adil, "TRAFFICKING AND SECRETION OF IL-10 FUSIONS IN TOBACCO BRIGHT YELLOW 2 CELL SUSPENSION CULTURES" (2008). Digitized Theses. 4373.
https://ir.lib.uwo.ca/digitizedtheses/4373