MLKL is considered the executor of necroptosis, a form of programmed necrotic cell death that occurs when caspase activity is inhibited. By studying the structure of the N-terminal four-helix bundle of MLKL, we found that helixes H2 and H5 are not tightly packed and are therefore most suitable for introducing mutations and consequently a disulfide bond. Disulfide bond could hinder the opening of the N-terminal four-helix bundle, that is likely necessary for the effector function of MLKL, thereby preventing either oligomerization or the insertion of the protein into the membrane. We mutated Leu at position 38 and Trp at position 109 into cysteines. To execute this mutations, we used pE-SUMOpro Amp vector with inserted opt1 (MLKL1 that has codon optimized for E. coli). Both mutations were successful, as we confirmed by nucleotide sequencing. We inserted the SUMO-optMLKL1 sequence, with both mutations, into the expression vector pET22b-pelB using IVA-cloning. NaDS-PAGE analysis after the expression of the fusion protein in bacterial cells of E. coli strains BL21[DE3] and BL21 [DE3]pLysS indicates that the protein is in the insoluble fraction. However, after the expression in BL21 [DE3]pLysS strain at 18°C, we found some protein to be soluble.
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