Cleaving pre-fusion state SARS-CoV-2 spike protein
Disclosed is producing recombinant SARS-CoV-2 spike protein in a pre-fusion state, using furin knock out or knockdown mammalian cells (such as HEK293, CHO or other mammalian cells) and using them to generate antibodies and related binding agents. The antibodies/binding agents can be used in SARS-CoV-2 detection assays or in diagnosis of active or prior infection with SARS-CoV-2; in prophylaxis or as a therapeutic; or for prophylactic or therapeutic use against coronaviruses related to SARS-CoV-2.
1. A method of generating the mature, active form of a SARS-CoV-2 S protein, comprising:
generating host cells which lack the ability to express furin but which express SARS-CoV-2 spike protein using a CRISPR Cas9 transfection protocol having sgRNA scaffolds: GATGCGCACAGCCCACGTGT (SEQ ID NO:19); and ACAGTGTGGCACGGAAGCAT (SEQ ID NO:20);
expressing the SARS-CoV-2 spike protein in said host cells; and
cleaving the SARS-CoV-2 spike protein with furin at a cleavage site between amino acid Arginine 670 and amino acid Serine 671 in the extracellular domain shown in SEQ ID NO.: 16, to yield the mature, active form of SARS Cov2 S protein containing the 51 and S2 subunits.