creator Zhou, Yi 2020 Spring 2020 Thesis Illinois Institute of Technology BIOL / Biology advisor Xiang, Jialing Biochemistry Molecular biology Cellular biology apoptosis BaxΔ2 cancer histidine tag protein purification recombinant protein en BaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from Baxα to induce apoptosis. The current computationally predicted model of BaxΔ2 is based on known Baxα structure, which is considered biased. Therefore, the elucidation of the BaxΔ2 crystal structure is critical. The goal of this project was to obtain a sufficient amount of purified recombinant Bax∆2 protein for crystallization. We cloned full-length BaxΔ2 fused with a poly-histidine tag on either N-terminus (His-Bax∆2) or C-terminus (Bax∆2-His) into an inducible bacterial expression vector. We found that His-Bax∆2 proteins were expressed better than Bax∆2-His, which totally inhibit host growth. However, the protein concentration of His-Bax∆2 was still too low to be detected by Coomassie blue staining. To increase His-Bax∆2 expression and avoid cytotoxicity, we further tested different bacterial host cells and applied the chaperone system. However, all attempts could not overcome Bax∆2 cytotoxicity and the protein expression levels were not high enough to be feasible for further large-scale purification. The mechanism underlying how Bax∆2 inhibits bacterial growth is still a mystery because Bax∆2 eukaryotic targets (mitochondria and caspases) do not exist in bacteria. Further experiments are required to explore the mechanism of Bax∆2 cytotoxicity in bacteria, so as to finally optimize and elevate the BaxΔ2 protein yields. born digital application/pdf In Copyright http://rightsstatements.org/page/InC/1.0/ Restricted Access http://hdl.handle.net/10560/islandora:1025029