ADAM17 is one of the key metalloproteinases in the human body. It is a transmembrane sheddase that facilitates the cleavage of the ectodomain of more than 90 substrates in the extracellular space, including the protein EpCAM. EpCAM can exist as a monomer or as a cis-homodimer. ADAM17 can cleave the monomeric form of EpCAM but not the dimeric form, as the cleavage site on EpCAM is inaccessible in such a complex. ADAM17 also exhibits adhesive properties. The mature form of ADAM17, generated upon prodomain removal, comprises six domains. Its extracellular region (ADAM17-EX) consists of the catalytic domain, the disintegrin domain, the membrane-proximal domain, and the CANDIS region. The regulation of ADAM17 activity is a complex process that involves interactions of this protein with various binding partners and the cell membrane, as well as conformational changes in its domains and interactions between them. To gain structural insights into the contribution of individual ADAM17 domains to its activity, we examined the extracellular region of ADAM17 and three truncated variants: CAT (containing the catalytic domain), CAT-DIS (containing the catalytic and disintegrin domains), and CAT-DIS-MPD (containing the catalytic, disintegrin, and membrane-proximal domains). We expressed and purified the CAT and CAT-DIS constructs. To improve the selectivity of purification for constructs containing the catalytic domain of ADAM17, we attempted to establish a method of specific affinity chromatography, using the ADAM17 prodomain as the stationary phase. We tested the chromatography column with the ADAM17-EX construct, but successful elution was not achieved. Finally, using the purified CAT construct, we analysed the cleavage of the EpCAM extracellular domain, both its monomeric and dimeric forms. Cleavage of neither form was successful.
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