an Ni:NTA resin. The concentration of SDS and urea tolerated by the resin is given by the supplier.
Also, it is essential to work at room temperature, because both urea and SDS can crystallize in the cold
room.
1. Isolate the inclusion bodies by differential centrifugations and determine the concentration of
protein by the BCA assay.
2. Prepare a solubilization buffer containing the appropriate denaturing agent. For example, for
α-helical MPs, the buffer contains 10 mM Tris/HCl pH 7.5, 100 mM NaH 2 PO 4 , 6 M urea,
0.8% SDS, 10% glycerol, and 4 mM β-mercaptoethanol (adapted from protocols described in
Banères et al. 2005; Damian et al. 2006). For porins, the solubilization buffer contains 10 mM
borate pH 10.0, 8 M urea, and 2 mM EDTA (Pocanschi et al. 2006, 2013). Notes: the
presence of reducing agent is required only if cysteine residues are present. The solubility of
urea can be increased to 10 M by heating.
3. Dissolve the inclusion bodies in the appropriate solubilization buffer at a final concentration
of 10 gÁL
À1 , and incubate overnight at room temperature. Note: sonication pulses can be
applied to speed up solubilization.
4. Centrifuge the sample for 20 min at 20,000 Â g in order to remove insoluble material.
5. Proceed to the purification step. For purifying α-helical MPs on Ni:NTA resin, the buffers are
(i) equilibration buffer: 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 0.8% SDS, and 4 mM βmercaptoethanol; (ii) elution buffer: 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 0.8% SDS,
400 mM imidazole, and 4 mM β-mercaptoethanol; (iii) desalting buffer: 50 mM Tris/HCl pH
8.0, 0.8% SDS, and 4 mM β-mercaptoethanol. For purifying β-barrel MPs, SDS in each
buffer is replaced by 8 M urea.
6. Determine the concentration of protein by UV-absorbance or by the BCA assay.
6.4.2
Renaturation of a-Helical MPs in APols
This step consists in exchanging SDS for APols (Fig. 6.6A). The optimal MP/APol mass ratio must
be determined by carrying out folding tests with variable amounts of APols.
1. Distribute 0.25 mg of the MP to be folded in three Eppendorf tubes. Add increasing volumes
of APol – 5 μL, 12.5 μL, and 25 μL – from a stock solution at 100 gÁL
À1 in order to obtain
MP/APol mass ratios equal to 1:2, 1:5, and 1:10. Note that lipids generally help in the folding
process – cf. Dahmane et al. (2009, 2013). Their usefulness can be tested by supplying them
(e.g. soybean lipids) to the samples so that the APol/lipid mass ratio is 1:0.2. This ratio can be
optimized, as well as the nature of the lipids.
2. Mix and incubate the samples for 30 min at room temperature.
3. SDS is eliminated by precipitating the dodecyl sulfate (DS) with KCl added from a 4 M stock
solution so that the final concentration of KCl in the samples is equal to 150 mM plus the
concentration of SDS. For example, if the volume of the sample is 1 mL and the concentration
of SDS is 0.8% (28 mM), the final KCl concentration should be 178 mM. The volume of KCl
to add is thus 44.5 μL.
4. Incubate for 30 min at room temperature under vigorous stirring.
5. Centrifuge the samples for 5 min at the maximum speed of a benchtop centrifuge at 20
C.
6. Collect the supernatant and repeat the centrifugation step.
7. Measure the optical density of samples at 280 nm.
6.4 Protocol 6.1. Amphipol-Assisted Folding of Membrane Proteins
355
Also, it is essential to work at room temperature, because both urea and SDS can crystallize in the cold
room.
1. Isolate the inclusion bodies by differential centrifugations and determine the concentration of
protein by the BCA assay.
2. Prepare a solubilization buffer containing the appropriate denaturing agent. For example, for
α-helical MPs, the buffer contains 10 mM Tris/HCl pH 7.5, 100 mM NaH 2 PO 4 , 6 M urea,
0.8% SDS, 10% glycerol, and 4 mM β-mercaptoethanol (adapted from protocols described in
Banères et al. 2005; Damian et al. 2006). For porins, the solubilization buffer contains 10 mM
borate pH 10.0, 8 M urea, and 2 mM EDTA (Pocanschi et al. 2006, 2013). Notes: the
presence of reducing agent is required only if cysteine residues are present. The solubility of
urea can be increased to 10 M by heating.
3. Dissolve the inclusion bodies in the appropriate solubilization buffer at a final concentration
of 10 gÁL
À1 , and incubate overnight at room temperature. Note: sonication pulses can be
applied to speed up solubilization.
4. Centrifuge the sample for 20 min at 20,000 Â g in order to remove insoluble material.
5. Proceed to the purification step. For purifying α-helical MPs on Ni:NTA resin, the buffers are
(i) equilibration buffer: 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 0.8% SDS, and 4 mM βmercaptoethanol; (ii) elution buffer: 50 mM Tris/HCl pH 8.0, 300 mM NaCl, 0.8% SDS,
400 mM imidazole, and 4 mM β-mercaptoethanol; (iii) desalting buffer: 50 mM Tris/HCl pH
8.0, 0.8% SDS, and 4 mM β-mercaptoethanol. For purifying β-barrel MPs, SDS in each
buffer is replaced by 8 M urea.
6. Determine the concentration of protein by UV-absorbance or by the BCA assay.
6.4.2
Renaturation of a-Helical MPs in APols
This step consists in exchanging SDS for APols (Fig. 6.6A). The optimal MP/APol mass ratio must
be determined by carrying out folding tests with variable amounts of APols.
1. Distribute 0.25 mg of the MP to be folded in three Eppendorf tubes. Add increasing volumes
of APol – 5 μL, 12.5 μL, and 25 μL – from a stock solution at 100 gÁL
À1 in order to obtain
MP/APol mass ratios equal to 1:2, 1:5, and 1:10. Note that lipids generally help in the folding
process – cf. Dahmane et al. (2009, 2013). Their usefulness can be tested by supplying them
(e.g. soybean lipids) to the samples so that the APol/lipid mass ratio is 1:0.2. This ratio can be
optimized, as well as the nature of the lipids.
2. Mix and incubate the samples for 30 min at room temperature.
3. SDS is eliminated by precipitating the dodecyl sulfate (DS) with KCl added from a 4 M stock
solution so that the final concentration of KCl in the samples is equal to 150 mM plus the
concentration of SDS. For example, if the volume of the sample is 1 mL and the concentration
of SDS is 0.8% (28 mM), the final KCl concentration should be 178 mM. The volume of KCl
to add is thus 44.5 μL.
4. Incubate for 30 min at room temperature under vigorous stirring.
5. Centrifuge the samples for 5 min at the maximum speed of a benchtop centrifuge at 20
C.
6. Collect the supernatant and repeat the centrifugation step.
7. Measure the optical density of samples at 280 nm.
6.4 Protocol 6.1. Amphipol-Assisted Folding of Membrane Proteins
355