Fig. 1. Directed evolution to generate high affinity ACE2 in 293T cells.
a ACE2 mutant library was expressed in 293T cells and incubated with the RBD of SARS-CoV-2 fused to superfolder GFP (sfGFP). b Error-prone PCR amplification of ACE2 protease domain induced random mutations. Mutant library-transduced cells were incubated with the RBD-sfGFP. Top 0.05% population with high level of bound RBD-sfGFP was sorted and underwent DNA extraction, followed by next cycle mutagenesis. Cell sorting was conducted by gating on forward scatter (FSC)-H and FSC-A to exclude doublets, followed by gating on Alexa 647 for HA-ACE2 expression and sfGFP for RBD-binding. c The value of KD and IC50 against pseudovirus of SARS-CoV-2 and SARS-CoV-1. d Neutralization potency to authentic SARS-CoV-2 was analyzed in Vero6E/TMPRSS2 cells. Data are mean of n = 3 technical replicates.