Protein expression and purification

The COVID-19 virus RBD recombinant protein was expressed in E.coli as inclusion body and in Bac-to-Bac expression system as soluble protein, respectively. The inclusion body was refolded and further purified by Superdex 200 Hiload 16/60 column (GE Healthcare) in 20 mM Tris, pH 9.0, 150 mM NaCl. Human ACE2 protein was expressed in baculovirus expression system. The constructed pFastBac1 plasmids were transformed into DH10Bac competent cells to generate recombinant bacmids. The bacmids were transfected into sf9 cells to generate virus and then amplified the virus and infected High five cells for protein expression. Both the soluble COVID-19 virus RBD and hACE2 were purified by HisTrap HP column (GE Healthcare) and were further purified by size-exclusion chromatography with a Superdex 200 column (GE Healthcare) in 20 mM Tris, pH 8.0, 150 mM NaCl. 

The plasmids of B38 heavy chain and light chain were co-transfected into HEK293T cells to produce B38 IgG. The full-length of B38 were used in neutralization and animal experiments. B38 Fab were generated by papain digestion and further purified by Protein A column (GE Healthcare) . 

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