PMC:7443692 / 48615-50316 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/7443692","sourcedb":"PMC","sourceid":"7443692","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7443692","text":"To express a stabilized ectodomain of Spike protein, a synthetic gene encoding residues 1−1208 of SARS-CoV-2 Spike with the furin cleavage site (residues 682–685) replaced by a “GGSG” sequence, proline substitutions at residues 986 and 987, and a foldon trimerization motif followed by a C-terminal 6xHisTag was created and cloned into the mammalian expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg, MD). The expression construct was transiently transfected in HEK293F cells using polyethylenimine (Polysciences, Inc, Warrington, PA). Protein was purified from cell supernatants using Ni-NTA resin (QIAGEN, Germany), the eluted fractions containing S protein were pooled, concentrated, and further purified by gel filtration chromatography on a Superose 6 column (GE Healthcare). Negative stain electron microscopy (EM) analysis was performed as described (Shaik et al., 2019). Briefly, analysis was performed at room temperature with a magnification of 52,000x and a defocus value of 1.5 μm following low-dose procedures, using a Philips Tecnai F20 electron microscope (Thermo Fisher Scientific) equipped with a Gatan US4000 CCD camera and operated at voltage of 200 kV. The DNA fragment encoding human ACE2 (1-615) with a 6xHis tag at C terminus was synthesized by Genscript and cloned to the vector pCMV-IRES-puro. The expression construct was transfected in HEK293F cells using polyethylenimine. The medium was discarded and replaced with FreeStyle 293 medium after 6-8 h. After incubation in 37°C with 5.5% CO2 for 5 days, the supernatant was collected and loaded to Ni-NTA resin for purification. 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