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Wilson's disease is caused by mutations in atp7b, a gene that encodes a copper-transporting P1B-type ATPase, otherwise known as the Wilson protein. Over 300 different mutations cause Wilson's disease, and we focused on missense mutations occurring in the 650 amino acid N-terminus of the Wilson protein. This portion of the protein includes six metal-binding domains that function to azquire Cu(I) from the cytosol, prior to passing it through the ATP-driven membrane-spanning pump. Of particular interest are metal-binding domains 5 and 6 (WLN5-6) since we know that they are absolutely required for function of the enzyme. Several missense mutations are found in WLN5-6 and in order to optimize expression of deleterious mutants we prepared a fusion protein consisting of a thioredoxin protein followed by a HHHHHH sequence and then a TEV protease site just upstream of WLN5-6. The gene for WLN5-6 was cloned into the pET-32Xa/LIC vector following manufacturers protocol (EMD Biosciences), by amplifying the gene using forward primer and reverse primers that complement the ligation independent cloning (LIC) site. For the forward primer the TEV protease site was included between the LIC site sequence and the beginning codon for WLN5-6. The successful vector construct, pET32Xa-TEV-WLN5-6, was confirmed by DNA sequencing, and then transformed into BL21(DE3) E. coli cells for protein expression studies. A single colony was inoculated into LB containing ampicillin and after the O/D/ reached 0.65, 1 mM IPTG was added to induce protein production. SDS-PAGE confirmed high level fusion protein expression. The fusion protein was purified using HisTrap chromatography and then cleaved with TEV protease, to remove the thioredoxin fusion protein from WLN5-6. This vector provides a platform to integrate deleterious Wilson's disease-causing mutations into WLN5-6.
Weitzner, Sandra, "Functional Variants of Metal-Binding Domains 5 and 6 of the Human Wilson Protein" (2014). Honors Theses. 2473.
Honors Thesis-Open Access