The N-terminal domain of MLKL (mixed lineage kinase-domain like protein), which contains a four-helix bundle (4HB) is crucial for inducing membrane permeabilization and triggering necroptosis. This domain alone is sufficient to carry out the necroptotic process, with the C-terminal helix (H6) acting as a regulatory element of its effector function. By studying the interactions between various de novo designed protein binders and the N-terminal part of the MLKL protein, we identified the most suitable one and expressed it. The selected protein, named GGNB MLKL BIND 005, was introduced into the PMCSG7 M33 APEX2 expression vector by IVA cloning, and the success of the insert insertion was verified by determining the nucleotide sequence. By testing expression in E. coli strain BL21 [DE3]pLysS at different temperatures, we determined after SDS PAGE analysis of the samples, that our fusion protein is best expressed at 25 °C. We used these conditions for large-scale expression. We then successfully isolated the protein, removed the N-terminal hexahistidine tag, and performed immunodetection with primary mouse monoclonal antibodies against c-Myc and secondary HRP conjugated goat antibodies against mouse IgG and IgM. We couldn’t demonstrate binding of the fusion protein to MLKL-154 by dot blot assay.
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