Parameters such as temperature and Mg
2+ concentration can be optimized at this stage.
4. In order to verify the solubility of the MP produced, 10 μL samples are diluted with the same
volume of 10 mM Tris/HCl, pH 8.0 buffer, centrifuged for 10 min at 16,000 Â g at room
temperature, and the supernatant and pellet analyzed by Western blot after SDS-PAGE
(cf. Figs. 7.8 and 7.9).
• CFE Large-Scale Reaction
Once the concentration of NAPols to be used has been optimized, the reaction can be scaled up to
1 mL of lysate:
1. The NAPol solution is prepared directly in the CFE reaction buffer used to resuspend the
lyophilized components of the reaction (commercial system) or in the S30 buffer for the
homemade lysate (Proverbio et al. 2014), so as to reach the desired final volume (1 mL) in the
presence of the chosen concentration of NAPol. The solution is stirred overnight in the
presence of 1% of sodium azide to guarantee optimal solubilization.
2. The 1 mL reaction is carried out in the presence of 15 μg of plasmid for 20–30 h in the
ThermoMixer at the optimized temperature.
NAPols do not cross dialysis membranes and, therefore, their presence in the feeding chamber is not
required.
3. After CFE, the protein can be purified by IMAC, either in batch or using a prepacked column.
Again, it is not necessary to add NAPols in the purification buffers.
Protocol prepared by Francesca Zito on the basis of Bazzacco et al. (2012) and laboratory notes, with
comments added.
References
Abdine, A., Park, K.H., Warschawski, D.E. (2012) Cell-free membrane protein expression for solid-state NMR. Meth.
Mol. Biol. 831:85–109.
Arévalo, M.A., Tejedor, F., Polo, F., Ballesta, J.P. (1988) Protein components of the erythromycin binding site in
bacterial ribosomes. J. Biol. Chem. 263:58–63.
Bazzacco, P. (2009) Non-ionic amphipols: new tools for in vitro studies of membrane proteins. Validation and
development of biochemical and biophysical applications. Thèse de Doctorat, Université Paris-7, 176 p.
Bazzacco, P., Billon-Denis, E., Sharma, K.S., Catoire, L.J., Mary, S., Le Bon, C., Point, E., Banères, J.-L., Durand, G.,
Zito, F., Pucci, B., Popot, J.-L. (2012) Non-ionic homopolymeric amphipols: Application to membrane protein
folding, cell-free synthesis, and solution NMR. Biochemistry 51:1416–1430.
Blesneac, I., Ravaud, S., Juillan-Binard, C., Barret, L.A., Zoonens, M., Polidori, A., Miroux, B., Pucci, B., PebayPeyroula, E. (2012) Production of UCP1, a membrane protein from the inner mitochondrial membrane, using the cellfree expression system in the presence of a fluorinated surfactant. Biochim. Biophys. Acta 1818:798–805.
Bogdanov, M., Dowhan, W. (1998) Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late
step of the conformational maturation of the polytopic membrane protein lactose permease. EMBO J. 17:5255–5264.
Breyton, C., Gabel, F., Abla, M., Pierre, Y., Lebaupain, F., Durand, G., Popot, J.-L., Ebel, C., Pucci, B. (2009) Micellar
and biochemical properties of (hemi)fluorinated surfactants are controlled by the size of the polar head. Biophys. J.
97:1077–1086.
376
7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins
2+ concentration can be optimized at this stage.
4. In order to verify the solubility of the MP produced, 10 μL samples are diluted with the same
volume of 10 mM Tris/HCl, pH 8.0 buffer, centrifuged for 10 min at 16,000 Â g at room
temperature, and the supernatant and pellet analyzed by Western blot after SDS-PAGE
(cf. Figs. 7.8 and 7.9).
• CFE Large-Scale Reaction
Once the concentration of NAPols to be used has been optimized, the reaction can be scaled up to
1 mL of lysate:
1. The NAPol solution is prepared directly in the CFE reaction buffer used to resuspend the
lyophilized components of the reaction (commercial system) or in the S30 buffer for the
homemade lysate (Proverbio et al. 2014), so as to reach the desired final volume (1 mL) in the
presence of the chosen concentration of NAPol. The solution is stirred overnight in the
presence of 1% of sodium azide to guarantee optimal solubilization.
2. The 1 mL reaction is carried out in the presence of 15 μg of plasmid for 20–30 h in the
ThermoMixer at the optimized temperature.
NAPols do not cross dialysis membranes and, therefore, their presence in the feeding chamber is not
required.
3. After CFE, the protein can be purified by IMAC, either in batch or using a prepacked column.
Again, it is not necessary to add NAPols in the purification buffers.
Protocol prepared by Francesca Zito on the basis of Bazzacco et al. (2012) and laboratory notes, with
comments added.
References
Abdine, A., Park, K.H., Warschawski, D.E. (2012) Cell-free membrane protein expression for solid-state NMR. Meth.
Mol. Biol. 831:85–109.
Arévalo, M.A., Tejedor, F., Polo, F., Ballesta, J.P. (1988) Protein components of the erythromycin binding site in
bacterial ribosomes. J. Biol. Chem. 263:58–63.
Bazzacco, P. (2009) Non-ionic amphipols: new tools for in vitro studies of membrane proteins. Validation and
development of biochemical and biophysical applications. Thèse de Doctorat, Université Paris-7, 176 p.
Bazzacco, P., Billon-Denis, E., Sharma, K.S., Catoire, L.J., Mary, S., Le Bon, C., Point, E., Banères, J.-L., Durand, G.,
Zito, F., Pucci, B., Popot, J.-L. (2012) Non-ionic homopolymeric amphipols: Application to membrane protein
folding, cell-free synthesis, and solution NMR. Biochemistry 51:1416–1430.
Blesneac, I., Ravaud, S., Juillan-Binard, C., Barret, L.A., Zoonens, M., Polidori, A., Miroux, B., Pucci, B., PebayPeyroula, E. (2012) Production of UCP1, a membrane protein from the inner mitochondrial membrane, using the cellfree expression system in the presence of a fluorinated surfactant. Biochim. Biophys. Acta 1818:798–805.
Bogdanov, M., Dowhan, W. (1998) Phospholipid-assisted protein folding: phosphatidylethanolamine is required at a late
step of the conformational maturation of the polytopic membrane protein lactose permease. EMBO J. 17:5255–5264.
Breyton, C., Gabel, F., Abla, M., Pierre, Y., Lebaupain, F., Durand, G., Popot, J.-L., Ebel, C., Pucci, B. (2009) Micellar
and biochemical properties of (hemi)fluorinated surfactants are controlled by the size of the polar head. Biophys. J.
97:1077–1086.
376
7 Amphipol-Assisted Cell-Free Expression of Membrane Proteins