Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Biology

Supervisor

Dr. Sangeeta Dhaubhadel

Abstract

Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal based systems. The main technical challenge during this process is to produce sufficient level of proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of a human therapeutic protein interleukin (IL) -10 produced in transgenic tobacco leaves was found to be below the critical level, and is potentially due to degradation by tobacco cysteine proteases (CysPs). A total of 60 putative CysP genes were identified in tobacco and based on their expression in the leaf tissue, 10 candidate CysPs (CysP1-CysP10) were selected for further characterization. The effects of silencing and overexpressing these CysPs on IL-10 accumulation were examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by stably silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the controls. Moreover, CysP6 is localized to the Endoplasmic Reticulum (ER), suggesting that ER is the potential site of IL-10 degradation. Overall results suggest that CysP6 is involved in determining the yield of recombinant IL-10 in tobacco.

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