Fig. 1.

Construct design and expression optimization of the SARS CoV-2 Spike (S) protein. (A) Linear representation of the S protein primary structure and construct design. Full-length (FL) S protein, extracellular S protein with tPA signal peptide (tPA-S-WT) and extracellular S-2P protein with tPA (tPA-S-2P) or the original signal peptide (Ori-S-2P). NTD, N-terminal domain; RBD, receptor binding domain; SD1, subdomain 1, SD2, subdomain 2; FP Fusion peptide, HR1, heptad repeat 1, CH, central helix, CD, connector domain, HR2, heptad repeat 2; TM, transmembrane domain; tPA, tissue plasminogen activator; CT, cytoplasmic tail. (B) Construct map of pcDNA3.1, pVRC8400 and pTT5 vectors that were inserted with the tPA-S-2P gene at the multiple cloning site. (C) SDS-PAGE analysis of Ni-Sepharose excel-purified S-2P protein produced with different vectors. (D) SDS-PAGE and western blot analysis of purified S-2P from two constructs with different signal peptides. (E) SDS-PAGE and western blot analysis of purified tPA-S-2P and tPA-S-WT produced via three types of 293 cell lines. To make the western blot image more clearly, we down-adjusted the Gray coefficient of exposure parameter. Human convalescent serum was used for immunoblotting.