Date of Award
Master of Science
Dr. Rima Menassa
Traditional protein purification methods are costly at the industrial scale. As therapeutic peptides demonstrate their potency against human disease, a strong impetus towards commercialized production emphasizes the development of novel purification methods. A two-component system was developed in tobacco BY-2 cells that used an elastin-like polypeptide tag and a terminally-cleaving small molecule-dependent intein to purify a green fluorescent protein (GFP) fusion protein and remove the purification tag from it. The small molecule-dependent intein, originally developed in yeast, appeared to splice illegitimately in tobacco BY-2 cells, yielding non-functional GFP, and exhibiting significant uninduced splicing. Conversely, once the intein was modified to cleave either at the N- or C-terminus, cleavage only occurred in a controlled manner in the C-terminus fusion protein through pH reduction. Inverse transition cycling of the elastin-like polypeptide (ELP) purification tag concentrated rather than purified the target protein, suggesting an alternative economical purification tag is required for cell extract purification.
Hendy, Braedon, "Recombinant protein purification in plants utilizing small molecule-dependent inteins and elastin-like polypeptides" (2009). Digitized Theses. 3901.