DNA is highly susceptible to damage caused by endogenous and exogenous factors. DNA damage also includes certain covalent DNA–protein crosslinks (DPCs), which occur when proteins become irreversibly covalently bound to chromosomal DNA. A common form of these lesions are TOP1−DPC complexes, which arise from the covalent binding of topoisomerase I (TOP1) to DNA in the form of an enzyme–DNA intermediate. In mammals, the removal of DPCs involves the SPRTN protease and the homohexameric AAA+ ATPase p97 (also known as VCP or Cdc48 in yeast). Recently, its adaptor protein TEX264 was identified, which recognizes TOP1–DPC lesions and recruits p97 to the site of damage, where p97 unfolds the covalently bound TOP1 protein. The mechanism of action of TEX264 protein remains poorly understood. As part of this master’s thesis, we performed a biochemical and biophysical characterization of the TEX264 protein. Since its 3D structure has not yet been experimentally determined, we used AlphaFold 3 tool for structural prediction, which revealed a high proportion of intrinsically disordered regions and a hydrophobic N-terminal α-helix. Based on this, we designed and synthesized two variants of the protein: full-length TEX264 and a truncated variant, $^{∆30}$TEX264, lacking the first 30 amino acid residues. Neither variant could be obtained in a soluble form, therefore both were expressed as fusion proteins with maltose-binding protein (MBP). This approach enabled the production of soluble Δ30TEX264 protein. Subsequently, six purification processes of $^{∆30}$TEX264 were performed, during which the steps were progressively optimized. Modifications included adjusting the matrix volume in the first and second Ni-NTA Superflow affinity chromatography steps, as well as testing different matrix sizes for size-exclusion chromatography. We also evaluated the effect of one versus two steps of MBP affinity chromatography and the presence of 0.05% Tween-20 detergent in the buffers. Additionally, we analyzed the influence of TEV protease concentration on cleavage efficiency. Protein purification proved challenging, as TEV protease cleavage was inefficient. Consequently, the yield of the isolated protein was low, and the sample contained impurities. Nevertheless, we obtained sufficient amount of protein for circular dichroism (CD) spectroscopy and N-terminal sequencing. The CD spectrum indicated a secondary structure composed of both α-helices and β-structures, consistent with the AlphaFold 3 predicted model. N-terminal sequencing confirmed the correct amino acid sequence of the first four residues of $^{∆30}$TEX264 protein. In future studies, the $^{∆30}$TEX264 protein could serve as a starting point for investigating interactions with p97 protein and other adaptor proteins involved in DPC repair. Such studies would contribute to a better understanding of the mechanism of action of TEX264 protein in the repair of TOP1–DPC lesions.
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