Multitron standard shaking incubator

Cells were harvested during exponential growth, centrifuged for 5 min at 9283g in a GS-15R (Beckman Coulter, Krefeld, Germany), resuspended to OD 40 in 0.9% (w v⁻¹) NaCl containing 25% (w v^− 1) glycerol, and 1 mL aliquots were frozen to − 80 °C for storage.
For pre-cultivation, 50 mL CGXIIstd medium was incubated in a fourfold baffled 500 mL shake flask at 30 °C, 300 rpm and 25 mm shaking diameter in a Multitron Standard shaking incubator (Infors, Einsbach, Germany). Depending on the experiment timing, the inoculum from cryocultures was varied in such a way that at harvest sufficient concentrations of biomass were available without exceeding the maximum oxygen transfer capacity of the shake flask system (according to preliminary experiments, this was reached when an optical density of 20 was exceeded). The culture was harvested, centrifuged for 5 min at 9283g in a GS-15R and resuspended in 0.9% (w v⁻¹) NaCl to obtain a stock suspension for the subsequent main cultures.

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.