Author: Yasunori Watanabe; Joel D. Allen; Daniel Wrapp; Jason S. McLellan; Max Crispin
Title: Site-specific analysis of the SARS-CoV-2 glycan shield Document date: 2020_3_28
ID: 63j4qc7d_22
Snippet: To express the prefusion S ectodomain, a gene encoding residues 1−1208 of SARS-CoV-2 S (GenBank: MN908947) with proline substitutions at residues 986 and 987, a "GSAS" substitution at the furin cleavage site (residues 682-685), a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag was synthesized and cloned into the mammalian expression vector pαH. This expression vector was used to trans.....
Document: To express the prefusion S ectodomain, a gene encoding residues 1−1208 of SARS-CoV-2 S (GenBank: MN908947) with proline substitutions at residues 986 and 987, a "GSAS" substitution at the furin cleavage site (residues 682-685), a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag was synthesized and cloned into the mammalian expression vector pαH. This expression vector was used to transiently transfect FreeStyle293F cells (Thermo Fisher) using polyethylenimine. Protein was purified from filtered cell supernatants using StrepTactin resin (IBA) before being subjected to additional purification by size-exclusion chromatography using a Superose 6 10/300 column (GE Healthcare) in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3.
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