3 Methods
Once the proteins are solubilized from the isolated membranes, it is
important to work at 4
C and proceed quickly with the protein
purification to avoid protein aggregation and degradation.
3.1 Expression
of HTL
(SecY-SecE-SecG +
YidC-SecD-SecF
+ YajC)
1. Transform the pACEMBL HTL3 plasmid (Fig. 1a) into chemically competent or electrocompetent E. coli BL21 Star (DE3)
or E. coli C43λ(DE3) cells [3] and select for growth on LB agar
plates with ampicillin, chloramphenicol, and kanamycin at
37
C overnight.
2. Inoculate 100 mL preculture in LB medium supplemented
with 50 μg/mL ampicillin, 20 μg/mL chloramphenicol, and
30 μg/mL kanamycin with one colony from the LB agar plate
and grow at 37
C overnight by shaking at 180 rpm.
3. Inoculate 1 L of Terrific Broth with 50 μg/mL ampicillin,
20 μg/mL chloramphenicol, and 30 μg/mL kanamycin in a
5 L baffled Erlenmeyer flask with approximately 20 mL preculture to reach an initial optical density (OD 600nm ) of 0.05 at
600 nm. Shake the E. coli cultures at 37
C and 150 rpm.
Typical culture volumes for holo-translocon preparations are
6–12 L.
4. When the cell cultures reach an OD 600 of 1.2–1.5 (mid-log
phase), induce protein expression by the addition of 21 mL of
10% L-arabinose (0.2% final concentration) and 0.5 mL of
1 M IPTG.
5. After 3 h of protein expression (37
C, 150 rpm), the cells are
harvested by centrifugation for 20 min at 5000 Â g (4
C) (see
Note 1).
6. Discard the supernatant and determine the weight of the cell
pellet (see Note 2).
3.2 Membrane
Preparation
1. Thaw ~10 g E. coli HTL3 cell pellet on ice.
2. Add 30 mL of HSGM buffer and one tablet EDTA-free protease inhibitor cocktail and 0.5 mM PMSF.
3. Resuspend the cells by pipetting.
4. Open the cells by two passages through a French press cell at
1000 psi (see Note 3).
5. Centrifuge the cell lysate at 16,000 Â g for 30 min (at 4
C).
6. Transfer the supernatant into an ultracentrifugation tube and
discard the pellet.
7. Centrifuge the cleared lysate for 2 h in a Ti70 rotor at
45,000 rpm (149,000 Â g) at 4
C.
Production of Multi-subunit Membrane Protein Complexes
11
Once the proteins are solubilized from the isolated membranes, it is
important to work at 4
C and proceed quickly with the protein
purification to avoid protein aggregation and degradation.
3.1 Expression
of HTL
(SecY-SecE-SecG +
YidC-SecD-SecF
+ YajC)
1. Transform the pACEMBL HTL3 plasmid (Fig. 1a) into chemically competent or electrocompetent E. coli BL21 Star (DE3)
or E. coli C43λ(DE3) cells [3] and select for growth on LB agar
plates with ampicillin, chloramphenicol, and kanamycin at
37
C overnight.
2. Inoculate 100 mL preculture in LB medium supplemented
with 50 μg/mL ampicillin, 20 μg/mL chloramphenicol, and
30 μg/mL kanamycin with one colony from the LB agar plate
and grow at 37
C overnight by shaking at 180 rpm.
3. Inoculate 1 L of Terrific Broth with 50 μg/mL ampicillin,
20 μg/mL chloramphenicol, and 30 μg/mL kanamycin in a
5 L baffled Erlenmeyer flask with approximately 20 mL preculture to reach an initial optical density (OD 600nm ) of 0.05 at
600 nm. Shake the E. coli cultures at 37
C and 150 rpm.
Typical culture volumes for holo-translocon preparations are
6–12 L.
4. When the cell cultures reach an OD 600 of 1.2–1.5 (mid-log
phase), induce protein expression by the addition of 21 mL of
10% L-arabinose (0.2% final concentration) and 0.5 mL of
1 M IPTG.
5. After 3 h of protein expression (37
C, 150 rpm), the cells are
harvested by centrifugation for 20 min at 5000 Â g (4
C) (see
Note 1).
6. Discard the supernatant and determine the weight of the cell
pellet (see Note 2).
3.2 Membrane
Preparation
1. Thaw ~10 g E. coli HTL3 cell pellet on ice.
2. Add 30 mL of HSGM buffer and one tablet EDTA-free protease inhibitor cocktail and 0.5 mM PMSF.
3. Resuspend the cells by pipetting.
4. Open the cells by two passages through a French press cell at
1000 psi (see Note 3).
5. Centrifuge the cell lysate at 16,000 Â g for 30 min (at 4
C).
6. Transfer the supernatant into an ultracentrifugation tube and
discard the pellet.
7. Centrifuge the cleared lysate for 2 h in a Ti70 rotor at
45,000 rpm (149,000 Â g) at 4
C.
Production of Multi-subunit Membrane Protein Complexes
11