Recombinant Protein Purification from E. coli

lsr2 genes were amplified from genomic DNA of the M. smegmatis mc² 155 and M. tuberculosis strains by polymerase chain reaction (PCR) using appropriate primer pairs (Tsingke Biotech, Beijing). The amplified DNA fragments were cloned into modified pET28a expression vector to obtain recombinant plasmids that were introduced into E. coli BL21 (DE3) cells. The cells were cultured to an optical density at 600 nm (OD₆₀₀) of 0.6 in 200 mL LB medium (Trytone 20 g/L (#LP0042B, OXOID), Yeast Extract 10 g/L (#LP0021B, OXOID), NaCl 20 g/L (#A501218, Sangon Biotech)). Protein expression was induced by addition of 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (#I8070, Solarbio) for 12 h at 16 °C. The cells were collected by centrifugation and the protein (His-tag at the C-terminal) was purified by affinity chromatography on Ni-NTA agarose (#SA05101L, Smart-Lifesciences)^{28 (link),36 (link)}. The column-bound protein was washed with a wash buffer (100 mM Tris–HCl (#T1503, Sigma) pH 8.0, 500 mM NaCl and 40 mM imidazole (#I8090, Solarbio)) and dialyzed using an elution buffer (100 mM Tris–HCl pH 8.0, 500 mM NaCl and 250 mM imidazole). The protein was stored at −80 °C. Purified proteins were verified by sodium dodecyl sulfate-polyacrylamide gel Electrophoresis (SDS-PAGE). Protein concentration was detected by the Bradford method and Nano Drop (Thermo Fisher, USA).