MSAD-1 Protein Purification from M. abscessus

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).