Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Dr. Shengwu Ma

Abstract

The world-wide demand for recombinant proteins continuously increases as new medical and industrial applications are developed. Higher plants have the potential to help meet this rising demand as green bioreactors. A major hurdle, however, is low recombinant protein yields in higher plants and, as with many production systems, high cost associated with downstream purification and the production of short peptides. The goal of this research was to address each of these problems to further increase the utility of plant bioreactors. The short antigenic peptide 277 (p277) from heat shock protein 60, which has the potential for use in the prevention of type 1 diabetes, was fused to an known adjuvant, the non-toxic B subunit of cholera toxin (CTB). The fused CTB-p277 retained the ability to form a homo-tetramer, and bound to GM1 ganglioside, allowing for the oral delivery of the fusion protein to induce mucosal tolerance and prevention of diabetes. Protein yield increases can be achieved by either increasing overall plant yield or increasing the relative accumulation of the desired protein. Using RNA interference, a knockdown of Solanum tuberosum plastidic ATP/ADP transporter, involved in energy transport into heterotrophic plastids, resulted in a 30% increase in tuber biomass, as well as a two-fold increase in soluble protein content. In addition, expression of a monoclonal antibody in the knockdown line produced double the concentration of antibody per soluble protein compared to wild-type. Taken together, this is a 4-fold yield increase compared to wild-type. Protein purification accounts for a significant portion of its production cost. We developed the recombinant production of soybean agglutinin (SBA) for potential use as an affinity tag. SBA was purified to high quality using an agarose-N-acetyl-D-galactosamine column, resulting in a 1-step purification process. Recombinant SBA performed identically to native SBA during in vitro assays, including agglutination of red blood cells (RBC). A fusion of SBA with green fluorescent protein (GFP) resulted in SBA-GFP that retained its in vivo fluorescence, purification through a 1-step process as well as the ability to agglutinate RBC and in vitro fluorescence of the agglutinated cells.


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