Expression and purification of His-tagged cytochrome bo3
from E. coli C43 strains
Stock solutions/materials:
Same as the materials needed for the purification from E. coli GO105 strain, with one
additional solution:
1 M IPTG
Protocol:
1) Inoculate a colony of transformed E. coli C43 wild-type bo3/E286C strain in
200 ml of LB|0.1 mg/mL carbenicillin broth (add 200 μL 100mg/mL car. into
200 mL); incubate overnight at 37 °C|200 RPM.
2) Prepare 8*500 mL of LB broth|0.1 mM CuSO4|0.1 mg/mL carbenicillin (add
500 μL 100 mM CuSO4 and 500 μl 100mg/mL car. into 500 mL in a 2 L
baffled flask); sterilise, measure OD and inoculate them with 2 % starter
culture (10 mL inoculums into 500 mL).
3) Incubate at 37°C/200 RPM until OD600 reaches 0.6; don’t forget to subtract
LB’s OD; monitor OD hourly.
4) Add IPTG to a final concentration of 0.4 mM (200 µL of 1 M stock into 500
mL).
5) Continue the incubation for a further 3 h.
After this step, follow the same procedures in the purification from E. coli GO105
strain.