Expression and Purification of hOAT Variants

hOAT variants were expressed in E. coli and the cell lysate was treated as previously described (Montioli et al., 2017 (link)). The soluble fraction was loaded on a DEAE Sepharose 26/20 equilibrated with 20 mM sodium phosphate buffer, pH 7.6. A linear gradient from 20 to 180 mM sodium phosphate buffer, pH 7.6, was then applied. Under these conditions, OAT wild-type and all the pathogenic variants elute from the column between 110 and 160 mM sodium phosphate. Active fractions were then concentrated using an Amicon Ultra 15 unit (Millipore) and applied to a Superdex 200 XK 16/60 column (GE Healthcare) equilibrated in 50 mM HEPES, pH 7.4, NaCl 200 mM. Purified protein was concentrated and stored at −20°C. The purity of each preparation assessed by SDS-PAGE was >95%. The final yield of OAT WT, Q90E, R154L, G237D, R271K, E318K and C394Y variants were 35, 4, 26, 24, 7, 11 and 8 mg/L of liquid culture, respectively.