20-S2S-THs-F

SARS-CoV-2 recombinant Spike (SCoV2-rS) glycoprotein trimeric ectodomain from mammalian HEK293 cells, frozen

Description: SARS-CoV-2 recombinant Spike (SCoV2-rS) glycoprotein ectodomain expressed as secreted trimeric protein in mammalian (human) HEK293 cells. UniProtKB sequence accession number: P0DTC2 (SPIKE_SARS2); expressed ectodomain includes amino acids (aa) 1-1208. Whole expression construct includes: full-length SCoV2-S ectodomain (aa 1 – 1208) w/o transmembrane and cytoplasmic aa, furin cleavage site “RRAR” mutated to “GSAS”, C-terminal GCN4 trimerization motif fused to protein sequence, then follows thrombin cleavage site, Strep-tag II and His6-tag. Two mutations (K986P and V987P) were introduced into SCoV2-S sequence to stabilize the trimer in the pre-fusion conformation. SCoV2-rS glycoprotein was purified from culture medium under native (non-denaturing) conditions using convenient chromatographic techniques. Then it was dialyzed against PBS, adjusted to 1.0 mg/ml concentration, filter-sterilized, aliquoted and frozen for storage.

Form: Frozen liquid in PBS (pH 7.4).

Storage: The product is shipped with dry ice (shipment cost to Europe: 160 €). Store at -80°C for the long term. After thawing may be stored for short-term at 4°C (stable at least 2 weeks at this temperature). Avoid repeated freeze/thaw cycles.

Concentration: 1.0 mg/ml.

Purity: >90 % as determined by SDS-PAGE.

Image: SDS-PAGE showing SCoV2-rS product 20-S2S-THs-F at approximately 160 kDa (1, 3 and 5 μg/lane).

Molecular weight (MW): Predicted MW of SCoV2-rS is 142.11 kDa. Expression in mammalian cells ensures authentic post-translational modifications including glycosylation. Glycosylation increases MW, therefore SCoV2-rS is detected as intact protein at ~160 kDa (from CHO cells) or at 160-200 kDa (from HEK293 cells), respectively. The observed differences in glycosylation extent did not result in significant differences in anti-SARS-CoV-2 IgG ELISA assay.

Applications (tested): ELISA, Western blot, SDS-PAGE.

Quality: According to the expressed construct, purified SCoV2-rS protein should be trimer in the stabilized prefusion conformation. Full-length trimeric protein enables detection of more antibodies against SCoV2-S, than shorter protein constructs (e.g., RBD or S1). This product 20-S2S-THs-F showed similar results in ELISA with COVID-19 positive and negative patient sera, as analogous protein construct derived from CHO cells (see product 20-S2S-TCg-G information), but a lesser number of individual sera was tested. SCoV2-rS from HEK293 cells was not selected for further applications in broader population studies, because analogous product from CHO cells seems more convenient (due to more uniform glycosylation and higher yields).

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