Cell death is a key biological process for maintaining homeostasis and defending the organism against infected, mutated or damaged cells. In the mentioned cases, the cell can trigger a signaling response that leads to apoptosis, controlled cell death, or to necrosis, uncontrolled cell death. In the last decade, much attention has been drawn to necroptosis, which is considered to be a somewhat controlled process followed by pathological conditions that are not controlled, such as inflammation. The main mediator of the signal response in necroptosis is MLKL (mixed-lineage kinase domain-like), which consists of a pseudokinase domain and a four-helix bundle (4HB) domain. Studies have shown that the 4HB domain is responsible for the oligomerization and translocation of MLKL to the cell membrane, where it causes its permeabilization. In the Master's thesis, we wanted to check whether a shortened part of MLKL, which consists of 123 amino acid residues and represents the 4HB domain, compensates for cell death. We successfully expressed MLKL123 in the bacterial expression strain E. coli BL21 pLysS and isolated it using affinity chromatography, but problems arose during the isolation, as in addition to the bands that represented our protein, some other non-specific bands appeared (potential degradation expression products). We also successfully prepared two stable lines in the mammalian cell line HEK FlpIn-293, one containing only MLKL123, and the other containing, in addition to the protein, the EGFP fusion protein, which served for easier observation with a fluorescence confocal microscope. In both cases, we found that the protein was mainly located in the area of the cell membrane, but the MTT test showed that none of the constructs showed cytotoxicity, despite the round morphology of the cells.
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