Author: Dhurvas Chandrasekaran Dinesh; Dominika Chalupska; Jan Silhan; Vaclav Veverka; Evzen Boura
Title: Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein Document date: 2020_4_5
ID: ezpjdz55_13
Snippet: Protein expression, and purification -N-terminal domain of the SARS CoV-2 N protein (N-NTD, residues 44-180) was expressed as a fusion protein with 6xHis tag followed by cleavage site for TEV protease on its N-terminus. Escherichia coli BL21(DE3) expressing the protein were grown in minimal media containing 15 NH4Cl and [U-13 C]glucose. The culture was lysed by sonication in lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole, 10% glyce.....
Document: Protein expression, and purification -N-terminal domain of the SARS CoV-2 N protein (N-NTD, residues 44-180) was expressed as a fusion protein with 6xHis tag followed by cleavage site for TEV protease on its N-terminus. Escherichia coli BL21(DE3) expressing the protein were grown in minimal media containing 15 NH4Cl and [U-13 C]glucose. The culture was lysed by sonication in lysis buffer (50 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole, 10% glycerol, 3mM β-mercaptoethanol) and the lysate was cleared by centrifugation. His-tagged protein was purified from the supernatant by affinity chromatography on a Nickel-NTA column (Machery-Nagel) according to manufactur's instruction, the 6x His tag was cut off by the TEV protease and the protein was further purified by size-exclusion chromatography on a Superdex 75 HiLoad 26/60 column (GE Healthcare) in SEC buffer (20 mM Na2HPO4, 50 mM NaCl, 0.01% NaN3, pH 5.5). Purity of the protein was checked using SDS-PAGE. Protein was concentrated to 1.19 mM and used for NMR experiments. For further NMR measurements of binding RNA, the N-NTD was diluted to 300 μM and flash frozen in liquid nitrogen and stored in -80°C until needed.
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