Protein purification

The antitoxin gene Aave_1719 was transferred to E. coli BL21 (DE3) (Tsingke, Beijing, China) and a pET28a vector that contained a GST-tag for expression of the Aave_1719 protein. During protein expression, the culture was initially grown overnight in LB broth supplemented with 50 µg mL⁻¹ kanamycin, before 150 mL 1:100 subcultures were prepared in 250-mL flasks, and grown at 37°C with shaking at 200 rpm until reaching an OD₆₀₀ of 0.6–0.8. Protein expression itself was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cultures were subsequently incubated for a further 6 hours at 28°C with shaking at 150 rpm. However, the toxin gene Aave_1720 was expressed in E. coli Top10 competent cells (Tsingke, Beijing, China) via the pBAD vector, which contained a His-tag. The strain was grown overnight with shaking in LB broth containing 50 µg mL⁻¹ ampicillin and 0.2% (wt/vol) glucose and then inoculated at a 1:100 dilution into 150 mL LB broth supplemented with 50 µg mL⁻¹ ampicillin and 0.2% (wt/vol) glucose for further cultivation. The culture was incubated at 37°C with shaking at 200 rpm until reaching an OD₆₀₀ of 0.6–0.8, followed by centrifugation at 5,000 × g for 10 min at 4°C to collect the cell pellet. The cell pellet was resuspended in 150 mL LB broth supplemented with 50 µg mL⁻¹ ampicillin and 0.2% (wt/vol) arabinose, and the induction of toxin protein Aave_1720 was performed at 28°C with shaking at 150 rpm for a further 6 hours. To purify the protein, the cells were pelleted by centrifugation at 5,000 × g for 10 min at 4°C and resuspended in 15 mL Lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1× protease inhibitor cocktail), with the buffer of the His-tagged proteins being supplemented with 10 mM imidazole. The cell walls were then disrupted by ultrasonication on ice, and the cell debris removed by centrifugation at 4°C. The resulting supernatants were loaded on Ni-NTA (BIORIGIN, Beijing, China) and glutathione resins (LABLEAD, Beijing, China) for the His-tagged and GST-tagged proteins, respectively. The bound protein was subsequently eluted with 1 mL elution buffer (50 mM Tris pH 8, 150 mM NaCl) containing 250 mM imidazole in the case of the His-tagged proteins and 10 mM reduced glutathione for the GST-tagged proteins. The resulting elution fractions were then concentrated before the proteins were harvested and transferred to storage buffer (50 mM Tris pH 8, 150 mM NaCl) using an Amicon Ultra Centrifugal Filter Unit (Merck Millipore, MA, USA). All samples were stored at −80°C until being required for further analysis.