I would like to reconstitute a 2x40 kDa membrane protein into liposomes and to make transport assays. In the literature, such protocols are given and often include pelleting of proteoliposomes for washing and medium exchange. Now, the centrifugal forces given vary among the different sources. For example "ultracentrifugation at 140,000 g for 45 min to pellet proteoliposomes", but also "before reconstitution the aggregated protein was removed by centrifuging the sample at 100,000 g for 30 min and protein concentration was measured at 280 nm" or "The KcsA-DOPE/DOPS proteoliposomes precipitate within a few hours and are visible as a cloudy white precipitate inside the dialysis tube... The liposome pellet was then harvested by spinning at 5500 g for 30 min." This looks like pelleting a precipitate.
Can anybody contribute her/his experience with such systems, especially if one would like to separate protein precipitates formed during the reconstitution procedure from proteoliposomes?
There are several different steps involved.
First you can pellet proteoliposomes, e. g. at 140,000 g for 45 min, to isolate them from soluble proteins.
Then you solubilize the membrane proteins with a detergent to bring them into solution. For safety you can now remove some aggregated fraction at e. g. 100,000 g for 30 min, as the solubilized membrane proteins should stay in solution. Most aggregates would also be removed at slower spin and shorter times, e. g. 16,000 g for few minutes (depending on sample size). The supernatant contains the protein to be reconstituted into proteoliposomes afterwards.
Now you can take the supernatant and reconstitute the protein into liposomes. It would be much more difficult to separate aggregates from already reconstituted protein in proteoliposomes, as these are large in size, and depending on conditions might sediment themselves, as you describie. So always remove the aggregates as long as the membrane proteins are still solubilized.
Btw: 100,000 g and 140,000 g are not necessarily very different in practise. These forces are able to pellet smaller aggregates, but keep soluble monomeric proteins in solution.. and that is what you want to achieve.
Thank you Andreas
Well, when we prepare liposomes with a very high protein/lipid ratio, we observe something that I would call precipitate (the solution is not clear any more). This corresponds exactly to what was described in one of the papers I cited - curiously, they however study this whitish object that sediments quite fast by solid state NMR and obtain quite reasonable spectra. I wondered whether during liposome reconstitution, protein may aggregate (as it cannot enter the membrane which may be saturated by too much protein and once the detergent is absorbed by the Biobeads ...). So maybe one could insert an additional centrifugation step at lower g to try to separate aggregates (that formed during the reconstitution process) from proteoliposomes. I'll try !
Yes, that's true!
If it sediments from alone, slow centrifugation speed should be enough to pellet it. And if something appears, maybe use a little lower protein/lipid ratio.. could improve it. Best!
I have had some good results reconstituting a detergent solubilized membrane protein into proteoliposomes by gel filtration, without using ultracentrifugation. The protein, detergent, and lipid are mixed together and passed over a Sephadex column that has no detergent in it. Some of the protein gets incorporated into the proteoliposomes when the detergent is diluted by the chromatography. Any that isn't probably gets stuck on the column (The Sephadex is thrown away afterwards). The proteoliposomes can be used immediately after eluting from the column. Elution of the proteoliposomes is visible due to the slight turbidity they impart.
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