Browsing by Author "Taha, Lamia"

The endoplasmic reticulum (ER) is an important organelle of the cell where a high number of proteins are synthesized and modified to obtain their final structure. Therefore, the ER stress, which is caused by accumulation of unfolded proteins in the ER, is not to be taken lightly since it could contribute to many diseases, such as cancer and neurodegenerative diseases. The response to the ER stress is the unfolded protein response (UPR), which is an adaptive system that helps in adjusting for increased folding needs within the ER. One of the main protein branches in the UPR is inositol requiring enzyme 1 (IRE1). IRE1 detects the status of protein folding inside the ER and initiates the UPR signaling pathway to achieve either normal folding status or cell death. The aim of this research was to express yeast IRE1 in E.coli and human IRE1 in insect cells, purify with affinity chromatography and study the IRE1’s crystal structure with a small molecule modulator that could possibly enhance its activity. The protein was expressed successfully and purified with glutathione S-transferase (GST) tag, and the activity of the pure protein was determined. The structural studies were not fully completed since the absolute purity and yield that was necessary for crystallization was not achieved due to loss of protein during gel filtration and precipitation. Based on the results it is likely that the structure of the protein could be solved and further biochemical and structural studies with F10 are possible.