Degree
Master of Science
Program
Biology
Supervisor
Dr. Rima Menassa
Abstract
Elastin-like polypeptides (ELP) and hydrophobins (HFBI) are fusion tags which enhance the accumulation levels of recombinant proteins in plants and aid in the non-chromatographic purification of these proteins. An intein is inserted between the target protein and fusion tag to replace the protease cleavage site. In this study, transient expression of intein-GFP fused to ELP or HFBI in Nicotiana benthamiana yielded the full size fusion protein. The ELP-fused proteins were successfully purified using inverse transition cycling (ITC) with 1.0 M NH4(SO4)2 at room temperature. However, the purification using membrane ITC was unsuccessful due to clogged membrane pores. Purification of HFBI-fused proteins through aqueous two-phase system (ATPS) using Triton X-114 was also unsuccessful. The proteins did not separate into the correct phase for recovery. In crude extract, cleavage of the intein at the C-terminus could be induced with a pH change, while DTT was not sufficient for cleavage at the N-terminus.
Recommended Citation
Liu, Kira, "Purification of Recombinant Proteins in Plants Using Small-Molecule Dependent Inteins Fused to ELP or HFBI" (2015). Electronic Thesis and Dissertation Repository. 3048.
https://ir.lib.uwo.ca/etd/3048