The disintegrin metalloprotease TACE or ADAM17 is an enzyme that functions as a sheddase and specifically cleaves the precursor tumor necrosis factor alpha. The enzyme also plays an important role in processing cell adhesion proteins, cytokine receptors and ligands of EGF receptors, that are needed for the stimulation of the immune response and regeneration. The extracellular multidomain region of TACE consist of: a prodomain, a catalytic domain, a disintegrin-like domain, a membrane-proximal domain and a CANDIS region. The prodomain acts as an inhibitor of TACE and was the focal point of our research work.
The preparation of a natively folded and stable prodomain was difficult for quite some time until scientists made a double mutant prodomain (TPD-DM) with R58A and C184A mutations.As part of our research work we tried to prepare a large enough amount of such prodomain to be used for the preparation of a column that is specific for the purification of the TACE enzyme using affinity chromatography. In this way we could facilitate and speed up the process of purification of the TACE enzyme.
We were able to prepare the genetic material for two TPD-DM constructs that had a hexahistidine tag added to the N-terminus to facilitate protein purification, and one of the
constructs also had a TEV protease cleavage region that enabled the removal of the
hexahistidine tag. After successful expression and purification of proteins by IMAC column
and dialysis, our proteins precipitated, making it impossible for us to isolate the proteins for
binding to the TACE specific purification column. We tried to optimize the elution, by reducing the concentration of imidazole in the elution buffer, as well as dialysis, to maintain the same salt concentration in the dialysis buffer as it was in the elution buffer but without much success. However we found out that cleavage of the hexahistidine tag most likely increases protein precipitation.
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