pCEP4-mCEACAM:Fc was constructed previously (78 (link)). Additional constructs were generated using the strategy described in reference 42 (link). Briefly, the mCEACAM coding region was removed by NotI and MreI digestion and replaced with the SARS-CoV-2 S NTD (codons 1 to 309), SARS-CoV-2 NTD-2/1, SARS-CoV-2 S RBD (codons 1 to 24 from the hCD5 signal sequence followed by SARS-CoV-2 S codons 310 to 529), or hACE2 ectodomain (codons 1 to 740). The expression plasmids were LipoD transfected into HEK293T cells, and transfected cells were incubated in FBS-free DMEM containing 2% (wt/vol) Cell Boost 5 (HyClone). Conditioned media were collected on days 4 and 7 and clarified free of debris (300 × g, 4°C, 10 min; 4,500 × g, 4°C, 10 min), and Fc-tagged proteins were then purified using HiTrap protein A high-performance columns (GE Healthcare) according to the manufacturer’s instructions. Purified proteins were dialyzed in PBS (pH 7.4), quantified spectrophotometrically, and stored at −20°C until use.
Free full text: Click here
