2.6. Protein Production

The two recombinant plasmids were used to transform Rosetta™ (DE3) pLysS E. coli cells. For recombinant protein production, 100 mL of auto-induction medium containing kanamycin (50 μg/mL) and chloramphenicol (34 μg/mL) was inoculated (1/100 v/v) for each protein. Cultures were then incubated for 24 h at 25 °C in a Multitron Standard shaking incubator (INFORS-HT, Switzerland) (300 rpm). Cells were collected by centrifugation and resuspended in a volume of lysis buffer dependent on the final culture OD of the individual expression, which was then frozen at −80 °C overnight. Cells were thawed, and incubated with 10 µg/mL DNAse and 20 mM MgSO₄ for 30 min shaking at 24 °C. To ensure complete cell lysis, cells were sonicated (Soniprep 150, MSE, UK) for 5 min (power 15, 30 s ON/OFF cycles), and subsequently centrifuged for 30 min at 20,000× g. Proteins were then purified using Ni Sepharose 6 Fast Flow resin (GE Healthcare, Uppsala, Sweden) and eluted with 250 mM imidazole elution buffer (50 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0). Purified protein was dialyzed overnight against protein buffer (50 mM Tris, 300 mM NaCl, pH 8.0) in Thermo Scientific™ Slide-A-Lyzer™ 10K MWCO dialysis cups, prior to storage at −80 °C until further use.