Isopropyl β d 1 thiogalactopyranoside (iptg)

The coding sequence of VvWRKY37 without stop codon was cloned in-frame to the pET32a prokaryotic expression vectors (6HIS tag). The homologous recombination method was used to construct VvABF2-HIS and VvABI5-HIS. All gene-specific primer pairs were designed using online software CE design V1.04 (Vazyme, Nanjing, China) (Supplementary Table 1). The recombinant vectors were introduced into E. coli strain TransB/DE3 (TransGen, Beijing, China). Recombinant protein induction was carried out by adding 2-mM isopropyl β-D-1-thiogalactopyranoside (Solarbio, Beijing, China) in the bacterial culture at 37°C for 6 h or at 16°C overnight with constant shaking at 150 rpm. The recombinant protein was purified by ProteinIso^® Ni-NTA Resinkit (TransGen, Beijing, China) according to the manufacturer’s protocol. The empty prokaryotic proteins (6HIS) were also induced, purified, and then used as a control in EMSA assays.

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).

The plasmid recombinant k3 was transformed into E. coli BL21 (Invitrogen, Waltham, MA, USA) competent cells and cultured (220 rpm, 37 °C) in Luria broth medium containing kanamycin (50 μg/mL) for 12–14 h. Once the optical density (OD600) reached the range of 0.6 to 0.8, cellular induction was initiated by adding 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Solarbio, Beijing, China) and continued for 8 h at 37 °C. Boiling method was applied for both product supernatant and inclusion bodies as previously described [19 (link)]. The protein expressions were verified by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

All the constructs with pET–28a or pGEX–6p–1 vectors were transformed into E. coli BL21 competent cells (Transgen biotech, Beijing, China) and cultured at 37 °C in medium containing 50 µg/mL kanamycin or 100 µg/mL ampicillin. When the culture optical density at 600 nm (OD600) reached 0.4 to 0.6, the cells were induced using one mM isopropyl β–D–1–thiogalactopyranoside (IPTG; Solarbio, Beiing, China) for 5 h. The supernatant and inclusion bodies were collected by centrifugation and sonication and the protein expressions were verified by SDS–PAGE.