Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism

Qi Liu; Amita Gupta; Ayse Okesli-Armlovich; Wenjie Qiao; Curt R. Fischer; Mark Smith; Jan E. Carette; Michael C. Bassik; Chaitan Khosla

Cell pellets were thawed and resuspended in lysis buffer containing Tris (40 mM, pH 7.5), NaCl (10 mM), imidazole (10 mM), DTT (1 mM). Cells were lysed by sonication and centrifuged at 25,000g for 1 h. The supernatant was incubated with a slurry of Ni-NTA resin for 1h at 4 °C and loaded onto a column. The nickel column wash buffer was Tris (40 mM, pH 7.5), NaCl (10 mM), DTT (1 mM) and each wash step contained increasing concentrations of imidazole (10, 50, or 250 mM). After SDS-PAGE confirmation of the protein fractions, protein was further purified using fast protein liquid chromatography (FPLC) using buffer A (50 mM Tris-HCl pH 8, 1mM dithiothreitol (DTT), 10% glycerol) and elution buffer B (50 mM Tris-HCl pH 8, 1mM dithiothreitol (DTT), 10% glycerol with 500 mM NaCl) with changing gradient over a 20 minute from 2% to 100% Buffer B. FPLC eluents containing the protein was concentrated using Amicon centrifugal filter with 3 K cut-off and the enzyme was stored in storage buffer (50 mM Tris-HCl pH 7.5, 10% glycerol).