Purification and Characterization of SOD1 Variants

Expression and purification of SOD1 were conducted as previously published [22 (link)]. Briefly, EGy118ΔSOD1 yeast were transformed with a wild-type SOD1, SOD1^A4V, SOD1^G93A, SOD1^H46R, or SOD1^G85R YEp351 expression vector and grown at 30°C for 44 h. Cultures were centrifuged, lysed in a blender using 0.5 mm glass beads, and subjected to a 60% ammonium sulfate precipitation. Then, the sample was centrifuged, and the resulting supernatant was diluted to 2.0 M ammonium sulfate. The diluted sample was passed through a phenyl-sepharose 6 fast flow (high sub) hydrophobic interaction chromatography column (Cytiva Life Sciences, Marlborough, Massachusetts, USA) using a linearly decreasing salt gradient from high salt buffer (2.0 M ammonium sulfate, 50 mM potassium phosphate dibasic, 150 mM sodium chloride, 0.1 mM EDTA, 0.25 mM DTT (pH 7.0)) to low salt buffer (50 mM potassium phosphate dibasic, 150 mM sodium chloride, 0.1 mM EDTA, 0.25 mM DTT (pH 7.0)) over 300 mL. Fractions containing SOD1 eluted between 1.6 and 1.1 M ammonium sulfate and were confirmed with SDS-PAGE. These fractions were pooled and exchanged into low salt buffer (10 mM Tris (pH 8.0)). Pooled fractions were then passed through a Mono Q 10/100 anion exchange column (Cytiva Life Sciences, Marlborough, Massachusetts, USA) using a linearly increasing salt gradient from low salt buffer to high salt buffer (10 mM Tris (pH 8.0), 1 M sodium chloride) from 0% to 30%. SOD1 fractions were collected between 5% and 12% high salt buffer and were confirmed with SDS-PAGE, western blot, and Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS).