Histrap fast flow crude column

SepinA3n recombinant protein was produced as already described [69 (link)], with some modifications. A pET(11a) expression vector containing the C-terminally (6x) His-tagged murine SerpinA3n (Genetech) was used to transform E. Coli BL21 (DE3) pLysS cells. Cells were grown in Luria–Bertani medium at 25 °C in presence of ampicillin (100 µg/mL) until OD₆₀₀ = 1, and then protein production was induced with 0.1-mM isopropyl 1-thio-D-galactopyranoside for 21 h at 15 °C. Bacterial cell lysis was performed by sonication using 5 cycles of 60 s on and 60 s of rest in ice. Cell debris was discarded by centrifugation and the crude extract was collected as the supernatant. The recombinant SerpinA3n protein was purified using HisTrap™ Fast Flow Crude column (GE Healthcare) with ÄKTA pure system. After eluting with a linear imidazole gradient, the fractions containing purified SerpinA3n were pooled together, dialyzed in 10 mM Tris–HCl and 50 mM KCl pH 8.0, and concentrated using Amicon® Ultra-15 Centrifugal Filters with 30 kDa cutoff (Merk Millipore).

NE06 was grown overnight under anaerobic conditions at 37°C and Nbs were precipitated from overnight cultures by ammonium precipitation as described above. The ammonium precipitated proteins were further purified via IMAC using a 5 ml HisTrap Fast Flow Crude column (GE). The purified Nbs were tested for anti‐NetB activity using fresh chicken red blood cells as described above.