E coli bl21 star de3 strain

Recombinant C-terminally his-tagged SOD1, cloned in pET303 vector, was expressed in E. coli BL21 Star (DE3) strain (Invitrogen). Culture from stock was grown overnight in ZYM-5052 autoinduction media (Studier, 2005 (link)) at +37 °C. Cells were harvested by centrifugation for 30 min at 6 000 rpm (HeroLab), 4 °C. The culture was first homogenized mechanically and then sonicated (VS70/T probe, Bandelin) in 50 mM sodium phosphate, 100 mM NaCl pH 7.5 buffer. Cell debris was pelleted by centrifugation for 30 min at 18 000 rpm, 4 °C. The supernatant was mixed with pre-equilibrated, Ni ²⁺ ion loaded IMAC resin (GE Healthcare) and left at 4 °C to equilibrate. The resin was loaded into GE Healthcare HiScale 26/40 column, the column was connected to ÄKTApurifier chromatographic system and target protein eluted with 200 mM imidazole step gradient. ApoSOD1 was obtained as described (Chattopadhyay et al., 2008 (link)) with slight modifications. Briefly, SOD1 was dialysed in 100 mM acetate, 50 mM NaCl, 10 mM EDTA pH 3.8 buffer overnight; then in 100 mM acetate, 50 mM NaCl, 50 mM EDTA pH 3.8 buffer for 4 h and two times in 10 mM potassium phosphate pH 7.4 buffer. Dialysed protein was filtered through a 0.22 µm filter (Millipore) and stored at −80 °C.

The E. coli BL21 Star (DE3) strain (Invitrogen) was transformed with the pGEX-4T-1, pGEX-4T-1-Sch9 and pGEX-4T-1-Pkh1 plasmids according to standard transformation protocols. Transformant colonies were picked into 3 ml overnight pre-culture in LB-ampicillin. The whole pre-culture was used to inoculate 500 ml of LB-ampicillin. The cells were grown at 37°C till OD_600nm of about 0.8. Protein expression was induced by adding 0.8 mM IPTG and the cells were incubated at 18°C with 200 rpm overnight shaking. Cell pellets were harvested by centrifugation, washed with ice-cold PBS, flash frozen in liquid nitrogen and stored at −80°C till further processing.