Variable Macro X Domain of SARS-CoV-2 Retains the Ability to Bind ADP-ribose

David N. Frick; Rajdeep S. Virdi; Nemanja Vuksanovic; Narayan Dahal; Nicholas R Silvaggi

Protein Purification-Colonies of BL21(DE3) cells harboring the pET21-COVID-MacroX plasmid were used to inoculate 3 ml of LB medium containing 100 mg/ml ampicillin. The starter culture was incubated at 37 °C with shaking at 225 rpm. After the cells grew to an OD600 of 1.0, they were transferred to 1 liter of fresh medium containing ampicillin. After the cells reached an OD600 of 1.0 again, protein production was induced with 1 mM isopropyl-β-D-thiogalactoside. After growing 16 h at 23 °C, the cells were harvested by centrifugation at 4,000 rpm, 4°C. The resulting cell pellet was suspended in 25 mL of IMAC buffer (20 mM Tris pH 8, 0.5 M NaCl), sonicated on ice for five 1 min bursts, with 2 min rests between, and clarified by centrifugation at 10,000g for 30 min. The supernatant was loaded onto a 5 ml Ni-NTA column and the fractions were eluted with a step gradient from 5 to 500 mM imidazole. Fractions containing the macro X domain protein (5 ml total) were loaded on a 250 ml Sephacryl S300 gel filtration column and eluted with 10 mM MOPS, 150 mM NaCl. Concentration of the purified protein was determined by measuring absorbance at 260 nm using a molar extinction coefficient of 10,555 M -1 cm -1 , which was calculated with the ProtParam tool (https://web.expasy.org/protparam/).