Hitrap protein a high performance columns

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.

pCEP4-mCEACAM-Fc was constructed previously (31 (link)).
Splice overlap extension PCR was used to insert an MreI restriction site and GSGGGG linker
codons between the mCEACAM (codons 1 to 142) and the human IgG1 splice donor site. Using
this modified construct, the mCEACAM coding region was removed and replaced with MERS-S1A
(codons 1 to 357), MERS S1A (N222D), and MERS S1B (codons 1 to 24 from human CD5 [hCD5]
signal, followed by MERS S codons 358 to 588).
HEK293T cells were transfected using the PEI method and then incubated in FBS-free DMEM
containing 2% (wt/vol) Cell Boost 5 (HyClone). Supernatants were collected on days 4 and 7
and clarified by sequential centrifugation (300 × g,
4°C, 10 min; 4,500 × g, 4°C,
10 min). The Fc-tagged proteins were then purified using HiTrap protein A
high-performance columns (GE Healthcare) according to the manufacturer’s
instruction. The resulting purified proteins were quantified spectrophotometrically and
stored at –20°C until use.