Purification of hOAT Recombinant Variants

hOAT recombinant variants were purified from E. coli expression and subsequent cell lysis following the steps previously described [5 (link)]. The soluble fraction of the lysate was loaded on a DEAE Sepharose 26/20 equilibrated with 20 mM sodium phosphate buffer, pH 7.6. Then, a gradient from 20 to 200 mM sodium phosphate buffer, pH 7.6, was applied. Under these conditions, both hOAT wild-type and pathogenic variants eluted at a concentration of sodium phosphate between 110 and 160 mM. By using an Amicon Ultra 15 unit (Merck & Co, Rahway, NJ, USA), fractions containing the hOAT enzymes were concentrated and then loaded on a Superdex 200XK 16/60 column (GE Healthcare, Chicago, IL, USA) equilibrated in 50 mM HEPES pH 8.0, 200 mM NaCl. Purified proteins were finally concentrated and stored at −20 °C. The purity of each preparation assessed by SDS-PAGE was >95%.