Fig. 1: The development of robust cell surface-display platform for high-throughput screening of S variants. | Cell Discovery

Fig. 1: The development of robust cell surface-display platform for high-throughput screening of S variants.

From: High-throughput screening of spike variants uncovers the key residues that alter the affinity and antigenicity of SARS-CoV-2

Fig. 1

a The backbone of lentivirus plasmid, in which S variant was fused with mCherry via a T2A linker. The expression of S variant was driven by the constitutive strong EF1α promotor. b MFI of FITC in the WT-S and the Δfurin-S cell line, which were incubated with various concentrations of biotin-ACE2 (0, 0.1, 0.5, 1, 2.5, 5, 10, 20, 40, 60, 80, 100 nM). The non-linear regression curve (Sigmoidal, 4PL) was fitted and the bars represented SD, n = 2. c Western blotting analysis of membrane protein samples that were extracted from the WT-S, Δfurin-S, and blank HEK293T cell line, respectively. The primary antibody targeted the S1 subunit. The MW of S (S1 + S2) and S1 was migrated as ~230 kDa and ~129 kDa due to glycosylation, respectively. β-actin (~42 kDa) was used as the loading control. d The flowchart for high-throughput screening of S variants, from Step1 to Step 4. e The cell sorting graph, where the percentage of SI, SII, and SIII cells (red rectangle) were ensured < 10%, 1%–2%, and 5%–10%, respectively. The middle and right bottom panel represented the negative control (NC) of the 1st and 2nd screen (both showing a clear background), with no ACE2 or mAb incubation, respectively. f The time schedule from a new emerging pathogen protein to gain the efficient information of its threatening mutation.

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