pcDNA3.1 SARS-CoV-2 S D614G was a gift from Jeremy Luban (Addgene plasmid # 158075; http://n2t.net/addgene:158075 ; RRID: Addgene_158075). SARS-CoV-2 S D614G protein, RBDwt, RBDδ, and RBDο were produced in Expi293 cells (Thermo Fisher Scientific) and were purified using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described previously (24 (link), 25 (link)).
Briefly, Expi293 cells were cultured at 37 °C with 5% CO2 for five days after transfection of each plasmid encoding SARS-CoV-2 S D614G protein, RBDwt, RBDδ, or RBDο (BA1). The supernatant was collected and passed over the Ni-NTA agarose resin column three times. After washing with 100 mL of phosphate-buffered saline (PBS), the his-tagged protein was eluted by elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole). Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel.
Briefly, Expi293 cells were cultured at 37 °C with 5% CO2 for five days after transfection of each plasmid encoding SARS-CoV-2 S D614G protein, RBDwt, RBDδ, or RBDο (BA1). The supernatant was collected and passed over the Ni-NTA agarose resin column three times. After washing with 100 mL of phosphate-buffered saline (PBS), the his-tagged protein was eluted by elution buffer (pH8.0, 50 mM sodium phosphate, 300 mM NaCl, and 250 mM imidazole). Finally, samples were buffer-exchanged into pH 7.4 PBS using Amicon Ultra-4 (Merck Millipore, Burlington, MA, USA) spin columns with a 10 kDa cutoff. The purity of purified samples was assessed by 14% SDS-PAGE gel.
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