Protein Purification and Analysis

Primers to introduce mutations in the Cus1(290–350) and Hsh49 gene fragments were designed and used as described in García-Nafría et al. (2016) (link). Glutathione hexa-histidine Cus1(290–350)p constructs were coexpressed with Hsh49p as described above except that cells were grown at 15°C after induction. After harvesting, cells were frozen, thawed, and resuspended in Pulldown buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 5 mM 2-mercaptoethanol with one cOmplete EDTA-free protease inhibitor cocktail tablet [Roche] per 100 mL) then lysed by sonication. After centrifugation at 72,000g for 30 min at 4°C, 800 µL of supernatant was mixed with 10 µL 2 M imidazole-HCl, pH 7.4 and 30 µL Ni-NTA resin. After 2 h incubation with gentle mixing at 4°C, the resin was pelleted by centrifugation and washed twice with 300 µL Wash buffer (Pulldown buffer with 25 mM imidazole-HCl), then resuspended in 100 µL SDS-PAGE loading buffer, heated for 2 min at 90°C, and the protein released was analyzed by SDS-PAGE.