Histrap hp his tag protein purification columns

Recombinant MSAD-1 protein of M. abscessus (NCBI accession number OLT57519.1) were purified from Escherichia coli as previously described with minor modification (Jeong et al., 2022 (link)). Briefly, the DNA sequence of MSAD-1 was amplified from M. abscessus ATCC 19977^T using PCR with following primer sets (forward primer, 5′-TTT GGA TCC ATG CCA TTG GTG CGC ATC GAC CTC-3′; reverse primer, 5′-AAA AAG CTT GTG CGC CTG CGG CGG GCA C-3′), and cloned into pET-28a. The expression and purification of MSAD-1 were commercially commissioned by Bionics (Seoul, Republic of Korea). In detail, the protein expression was induced in E. coli Rosetta2 (DE3) strains (Novagen, WI, USA) transformed with pET28a-MSAD-1 by adding 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 26°C for 6 h. Cultured bacterial cells were harvested and sonicated for 30 cycles at 70% amplitude. After centrifuge, the supernatant was purified with HisTrap™ HP His tag protein purification columns (Cytiva, MA, USA) for Ni-NTA affinity chromatography via ÄKTA go system (Cytiva, MA, USA). Purified proteins were subjected to endotoxin removal using Pierce™ high-capacity endotoxin removal spin columns (Thermo Scientific, MA, USA) and quantified by Pierce™ chromogenic endotoxin quant kit (Thermo Scientific, MA, USA).

The expression and purification of RBD and ACE2-Fc were done as described in the previous report⁴⁴ . RBD containing His-tag was purified via the ÄktaPure liquid chromatography system using HisTrap HP His-tag protein purification columns (Cytiva, USA). The constructs for the RBD variants, except for the Omicron BA.1 and BA.2 were produced with KLD enzyme mix (New England Biolabs, USA) using RBD as the template. Omicron BA.1 and BA.2 (I332-E554)-coding genes were produced by Integrated DNA Technologies (Coralville, USA). Original RBD gene coding for the same region was replaced with the respective sequence using Gibson assembly (New England Biolabs, USA). The primers used are disclosed in Supplementary information (Supplementary Table 2). The expression and purification for the RBD variants were performed identically to the method used for RBD.