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

Uridine cytidine kinase 2 (UCK2) was cloned into the pET-21a(+) vector using NdeI and NotI as restriction sites. This resulted in C-terminally 6×His-tagged UCK2. The resulting plasmid was transformed into E. coli BL21(DE3) cells by electroporation, recovered in SOC at 37 °C for 1 h and plated onto LB agar plates with carbenicillin overnight at 37° C. A single, isolated colony was inoculated into 30 mL LB supplemented with 50 µg/mL carbenicillin and grown at 37°C with shaking for 15 h. The next day, these cells were used to inoculate 1 L autoclaved LB with 50 µg/mL carbenicillin. The flask was shaken at 37 ºC. When an optical density (OD600) of 0.7 was reached, protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The temperature was changed to 18 °C and flasks were shaken for an additional 15 h. The cell pellets were collected by spinning the media at 5000 rpm for 10 min. The pellets obtained were frozen by liquid nitrogen and stored in -80 °C for protein purification.