Isopropyl β d 1 thiogalactopyranoside (iptg)

The recombinant plasmid was transformed into E. coli BL21 expression host cells, followed by incubation at 37 °C in LB medium containing 100 mg/ml kanamycin for 3 h. At an optical density of 0.6 to 0.8 at 600 nm (OD₆₀₀), the cells were induced with 0.8 mM IPTG (Biosharp, Anhui, China), further cultured at 25 °C for another 12 h and then harvested.
For protein purification, cells were harvested by centrifugation at 7000 rpm for 10 min in a high-speed refrigerated centrifuge (Hitachi, Tokyo, Japan), resuspended in PBS (pH 7.5) and lysed by passing through a high-pressure homogenizer at 1000 Bar. After centrifugation at 10,000× rpm and 4 °C for 10 min, the supernatant was filtered through a 0.45 μm pore size filter and loaded onto glutathione sepharose beads (GE Healthcare, Uppsala, Sweden). The proteins were eluted with elution buffer (9.5 ml ddH₂O, 50 mM Tris-HCl, 10 mM L-glutathione reduced glutathione). The GST-tag on the N-terminus was cleaved using TEV (Tobacco Etch Virus) protease (Solarbio, Shanghai, China) at 4 °C for 8 h.

The recombinant plasmids, pET‐28a (+)‐att, pET‐28a (+)‐trap, and pET‐28a (+)‐eAls, were transformed into in Escherichia coli BL21 (DE3) (Tiangen) and were expressed the ATT, TRAP, and eAls proteins with 0.1 mM isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG, Biosharp) induction at 37°C for 4 h, respectively. Then, the bacterial cells were obtained by centrifugation and were ultrasonicated, and the suspension was acquired. The His‐tagged ATT, TRAP, and eAls proteins were purified by using His‐binding‐resin (Novagen) according to the manufacturer's instructions. These proteins were confirmed with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot. For Western blot, anti‐His tag monoclonal antibodies (mAbs) (Sigma) and horseradish peroxidase (HRP)‐conjugated goat antimouse IgG antibodies (Sigma) were used as the primary antibodies, the secondary antibodies, respectively.