Expression and purification of His-tagged cytochrome bo3
from E. coli GO105/pRhisA
Stock solutions/materials:
1) Growth media (LB):
10 g/L bacto-tryptone
5 g/L yeast extract
10 g/L NaCl
Optional: 1.5 % w/w agar
2) 100 mg/mL carbenicillin (stored at -80 °C)
3) 100 mM CuSO4
4) W1 buffer (adjust pH to 7.4):
20 mM MOPS
5 mM MgSO4
30 mM Na2SO4
5) 1 M MgSO4
6) 50 % glycerol
7) 100 mM PMSF (stored at -80 °C)
8) 10 % DDM (stored at -80 °C)
9) 5 M NaCl
10) 1 M imidazole (adjust pH to 8.0)
11) 1M Tris.Cl (adjust pH to 8.0)
12) Solubilisation buffer (adjust pH to 8.0):
20 mM Tris.Cl
5 mM MgSO4
300 mM NaCl
20 mM imidazol
10 % glycerol
1 % DDM
Method:
1) Inoculate a colony of GO105/pRhisA in 100 ml of LB|0.1 mg/mL carbenicillin
broth (add 100 μL 100mg/mL car. into 100 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 and inoculate them with 2 % starter culture (10 mL
inoculums into 500 mL).
3) Incubate at 37°C/200 RPM until OD600 reaches 2.0 (about 5-6 hours).
4) Harvest cells (7 k RCF for 10 min); measure wet weight of cells; store at 20 °C if necessary (thaw at RT under running water if you do this).
5) Wash cells with 100 mL ice-cooled W1 buffer.
6) Re-suspend cells in W1 buffer (0.25 g cells/mL); add PMSF to a final
concentration of 0.1 mM.
Note: from this point, take a sample at each step; if the pellet/supernatant from a
centrifugation step is not used for the following experiment, keep them for
analysis.
7) Disrupt cells by two passages through the cell disruptor at 35 k psi.
8) Centrifuge cells at 12 k RPM for 10 min using a JA 25.50 rotor and a lowspeed centrifuge to remove unbroken cells, outer membranes, cell wall and
any possible inclusion bodies.
9) Transfer the supernatant into an ultracentrifuge tube (Beckman polycarbonate
tubes; Ti 70/50.2 tubes); all ultracentrifuge tubes must be almost full before
use; fill the tube with W1 buffer if necessary; pellet the mixed membranes at
45 k RPM for 1 h at 4°C using a Ti50.2 rotor.
10) Re-suspend the membranes in W1 buffer.
11) Determine protein concentration of the membrane vesicles using BCA
method.
12) You may snap-freeze these vesicles and store at -80 °C until ready to use.
13) Re-suspend membrane vesicles to 3 - 4 mg protein/mL in the solubilisation
buffer.
14) Incubate at 4 °C for 1 h on a Spiramix.
15) Ultracentrifuge supernatant at 45 k RPM for 45 min at 4°C (Ti 50.2 or 70 rotor)
to remove non-solubilised protein or other cellular components.
16) Prepare and cool all solutions for the nickel sepharose affinity
chromatography in the fridge:
Equilibration buffer A
(20 mM imidazole)
Wash buffer B
(40 mM imidazole)
Elution buffer C
(200 mM imidazole)
Elution buffer D
(400 mM imidazole)
1 M Tris.Cl (pH 8)
1 M MgSO4
50% glycerol
1 M NaCl
1 M Imidazole.Cl (pH=8)
100 mM PMSF
10 % DDM
Water
Total volume
2.4
0.6
24
30
2.4
0.12
0.6
59.88
120
0.8
0.2
8
10
1.6
0.04
0.2
19.16
40
0.5
0.125
2.5
2.5
5
0.025
0.125
14.225
25
0.4
0.1
2
2
8
0.02
0.1
7.38
20
60*CV
20*CV 12.5*CV 10*CV
Final concentration
in mL
20 mM Tris.Cl (pH=8)
5 mM MgCl2
5 or 10% glycerol
250 or 100 mM NaCl
X mM imidazole (pH=8)
0.1 mM PMSF
0.05 % DDM
17) Prepare a bench column with 2 mL nickel sepharose (GE Healthcare Ni
Sepharose 6 Fast Flow).
18) Wash the column with 5 column volumes (CV) of MilliQ water.
19) Equilibrate column with 30 CV of Equilibration buffer A.
20) Apply the solubilised membranes to nickel column; collect the flow-through
(Flow 1).
21) Wash column with 10 CV of Equilibration buffer A, then 10 CV of Wash buffer
B; collect flow through after washes (Flows 2 and 3).
22) Elute cytochrome bo3 with 10 CV of Buffers C and 10 CV of Buffer D (Flow 4
and 5); most cytochrome bo3 should be in Flow 4/Buffer C.
23) Concentrate the protein by centrifugation in a 100 kDa MW cut-off column by
successive centrifugation for 1 - 5 min at about 5 k RCF; time and RCF
depend on the protein concentration and the type of column used; do not
exceed the maximum RCF specified by the manual of the column.
24) Determine the protein concentration of Flow 1 – 5 using both BCA method
and the Soret band at 408 nm; use Equilibration buffer A to dilute the sample
and fill up the quartz cell for Soret band measurement.
25) Determine the protein concentration of the samples taken during the
experiment using BCA method and run SDS-PAGE on the samples.