Biomedical Engineering Reference
In-Depth Information
4. When diSC
3
(5) fl uorescence has reached a stable value (~40-50 s)
add 3-5
l mitochondria, mix, and continue measurement
until the decrease in fl uorescence fi nishes.
5. Add 3
ʼ
l of 100× inhibitor mix to uncouple respiratory chain
complexes, and trace fl uorescent changes until the increase has
reached its maximum again (
see
Note 6
).
ʼ
Authentic cytochrome
b
2
reaches the intermembrane space (IMS)
of mitochondria via a “stop-transfer” mechanism that comprises
the insertion of N-terminal segments into the matrix followed by a
retranslocation and processing event in the IMS. The fusion pro-
teins described here consist of the N-terminal 167 residues of the
cytochrome b
2
precursor fused to the complete mouse DHFR [
9
,
10
]. The
b
2
-sequence encompasses the complete mitochondrial
targeting signal but lacks residues 47-65 of the intermembrane
spacing sorting signal (
see
Fig.
3a
). Thus, the resulting preprotein,
named b
2
(167)
∆
-DHFR, is targeted to the mitochondrial matrix
instead of being exported to the intermembrane space.
For isolation of the preprotein in high amounts,
E. coli
cells
expressing the construct are lysed and the protein is purifi ed using
cation-exchange chromatography. The protein is eluted by applica-
tion of a salt gradient (0-0.5 M NaCl).
3.3 Preparation
of Precursor Proteins
from E. coli
1. Grow
E. coli
cells expressing the fusion protein b
2
(167)
ʔ
-
DHFR at 37 °C in LB medium to an OD
600
of 0.8.
2. Add IPTG to a fi nal concentration of 1 mM, and continue
growth at 30 °C for additional 2-3 h.
3. Harvest cells by centrifugation at 3,500 ×
g
for 8 min.
4. Wash cell pellet by resuspension in pre-lysis buffer and cen-
trifugation as in
step 3
.
5. Resuspend cells in buffer A with 0.1 g cell pellet per ml lysis
buffer. Destabilize cell walls by freezing the suspension in liq-
uid N
2
and subsequent thawing at room temperature for two
times. Add lysozyme to a fi nal concentration of 1 mg/ml, and
0.1 mg/ml DNAse, and incubate on ice for 10 min.
6. Add 0.1 % (V/V) Triton X-100 and solubilize cells on ice for
10 min.
7. Disrupt cells by sonication on ice: 3 × 20 pulses (chill cells on
ice briefl y between pulse intervals), 40 % duty cycle, micro tip
setting 7.
8. Pellet cell debris by centrifugation at 15,000 ×
g
for 20 min.
Take samples of both pellet and supernatant (lysate) to analyze
lysis effi ciency via SDS-PAGE.
9. Pour crude cell lysate over a cellulose acetate fi lter membrane.
Take sample of fi ltered lysate for SDS-PAGE analysis.
