Expression and purification of recombinant proteins

The recombinant E. coli BL21 Star™ (DE3) host strains were individually cultured on LB agar plates supplemented with 100 µg/ml ampicillin for 16 h at 37 °C. Individual colonies were picked and subcultured separately in 25 ml LB broth (with 100 µg/ml ampicillin) overnight at 37 °C. The culture broths were centrifuged for 10 min at 16,000 g at 4 °C and the pellets re-suspended in 500 ml of fresh LB broth and then incubated at 37 °C until optical density (OD₆₀₀) reached 0.4. Expression was induced with 1 mM of Isopropyl b-d-1-thiogalactopyranoside (IPTG) for 5 h at 37 °C and monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% separating gel. Induced cells were harvested by centrifugation at 16,000 g for 10 min at 4 °C, and the obtained pellets were washed twice in PBS (pH 7.2). Pellets for each of the rSUB, rFER2 and rP0 proteins were separately re-suspended in lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100, pH 8.0), incubated for 1 h at 22 °C and lysed using a Vibra-Cell VCX 500–700 ultrasonic homogenizer (Sonics Inc., San Jose, CA, USA) at 5 cycles of 30 pulses for 30 s. The lysate was centrifuged at 16000 g for 10 min at 4 °C, following which the supernatant was filtered through a 0.45-µm porosity filter (MilliporeSigma, Burlington, MA, USA); the targeted rSUB, rFER2 and rP0 proteins were then purified by nickel affinity column chromatography (GenScript). Purification was performed in PBS, pH 7.3 (washing and binding buffer) and 10 mM Tris HCl (pH 8.0), 0.5 M NaCl and 20 mM imidazole (elution buffer). Fractions containing the eluted proteins were dialyzed in PBS (pH 7.2) for 12 h at 4 °C. Protein purity was verified by SDS-PAGE in a 16% separating gel under reduced conditions. The concentrations were determined according to the standard bovine serum albumin (BSA) using the Bradford assay [44]. The purified recombinant proteins were stored in a storage buffer (50 mM NaHCO₃, 150 mM NaCl, 10% glycerol, pH 10.0) at − 70 °C for further use.