21-S2S-TCg-F/B1

SARS-CoV-2 Biotinylated Spike glycoprotein trimeric ectodomain from mammalian CHO cells, frozen

Description: Biotinylated SARS-CoV-2 recombinant Spike (SCoV2-rS-Biotin) glycoprotein ectodomain expressed as secreted trimeric protein in mammalian (hamster) CHO 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 GSN4 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 as well, 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 biotinylated by chemical labeling via amines using validated method, buffer changed to PBS, 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: 0.65 mg/ml.

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

Image: SDS-PAGE showing SCoV2-rS-Biotin product 21-S2S-TCg-F/B1 (in lane B1) at approximately 160 kDa (3 μg/lane). Lane F shows sample of non-labeled SCoV2-rS product 20-S2S-TCg-F used for biotinylating (3 μg/lane), whereas lane M – molecular weight marker (ThermoFisher, Cat. No. #26614).

Applications (tested): ELISA, other immunoassays, 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). It provides a tool to develop highly sensitive immunoassays.

Labeling method and analysis of Biotin incorporation: Labeling of the protein with Biotin was performed by validated method using commercially available Biotinylation Kit. The HABA Assay was used for measuring the level of Biotin incorporation. The biotin to protein ratio in SCoV2-rS-Biotin product 21-S2S-TCg-F/B1 is between 3 and 4 (Lot dependent) as determined by the HABA assay.

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