A codon optimized expression construct corresponding to the processed protein Nsp9 from the Betacoronavirus SARS-CoV-2 (2019-nCoV; COVID-19) Wuhan-Hu-1 isolate (Genbank MN908947.3) was synthesized and inserted in the pET28a-TEV vector at the NdeI/NotI restriction enzyme sites by Genescript (Piscataway, NJ). The recombinant plasmid was then used to transform chemically competent Escherichia coli Rosetta BL21(DE3)pLyS cells (Novagen, Millipore Sigma, Burlington, MA) by a heat shock method. The expressed gene product contained a 22 amino acid extension, MGSSHHHHHHSSGENLYFQGHM- at the N-terminus of the native protein to enable protein purification by metal chelation chromatography (Choi et al.., 2011). The SSGCID internal ID for the SARS-CoV-2 Nsp9 construct is BewuA.18928.a.MX101 (www.ssgcid.org).

Approximately 70% deuterated, uniformly 15N-, 13C-labeled protein was obtained by growing the transformed cells (310 K) in 750 mL of minimal medium (Miller) containing ~ 70% 2H2O (v), 15NH4Cl (1 mg/mL) and D-[13C6]glucose (2.0 mg/mL), NaCl (50 µg/mL), MgSO4 (120 µg/mL), CaCl2 (11 µg/mL), Fe2Cl3 (10 ng/mL) and the antibiotics chloramphenicol (35 µg/mL) and kanamycin (34 µg/mL). When the cell culture reached an OD600 reading of ~ 0.8, it was transferred to a 298 K incubator and protein expression induced with isopropyl β-D-1-thiogalactopyranoside (0.026 µg/mL). Cells were harvested by mild centrifugation following overnight incubation and then frozen at 193 K. After thawing the frozen pellet, Nsp9 was purified with a conventional two-step protocol involving metal chelate affinity chromatography on a 20 mL Ni-Agarose 6 FastFlow column (GE Healthcare, Piscataway, NJ) followed by gel-filtration chromatography on a Superdex75 HiLoad 26/60 column (GE Healthcare, Piscataway, NJ). In addition to removing minor impurities, the latter step exchanged Nsp9 into the buffer used for the NMR studies: 100 mM NaCl, 20 mM Tris, 1.0 mM dithiothreitol, pH 7.0.

Nitrogen-15 labelled Nsp9 was prepared using an autoinduction protocol and the protein purified as described above. The concentrated protein was diluted 1:1 with a Tobacco Etch Virus (TEV) Buffer (50 mM TrisHCl, 150 mM NaCl, pH 7.8) to a final volume of approximately 1 mL (~ 10 mg/mL) and treated overnight at 278 K with 20 uL of TEV protease (0.5 ug/mL, prepared in house) to remove the N-terminal tag. The cleaved protein, which contained three N-terminal scar residues afterwards (GHM-), was purified by re-application on the size exclusion column. To assist amide assignments, residue-specific, 15N-labeled Leu, Val, Phe, Lys, and Met samples were prepared using a modified “Redfield-medium” (Arachchige et al. 2018) and the protein purified as described above.

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