Say I am treating a lipoprotein with a lipoprotein lipase that will hydrolyze the lipid portion, how can I then purify just the protein portion of the lipoprotein from the solution?
I've considered Hydrophobic Interaction chromatography, but wouldn't the protein portion have a hydrophobic component to it as well, seeing as it was bound to lipid? Will that protein bind to the resin as well?
Does anyone know of a highly efficient way to accomplish this?
Thanks in advance!
Hello Arlind,
chromatographic separation by hydrophobic interaction chromatography or reversed phase chromatography might indeed be useful here, as the lipids should interact more strongly with the hydrophobic matrix due to their higher nonpolar-to-polar ratio. As in every chromatography, you should optimize the eluent gradient for best separation results.
Alternatively, for removal of detergents from aqueous solutions, Bio-Beads can be used, which are hydrophobic particels for efficient scavenging of any hydrophobic components from your sample, and they should also work for lipids. In your case, I would take care of sufficiently high ionic strength (salt) in your sample buffer to select for the more pronounced hydrophobic interactions (between beads and lipids). The beads can be easily sedimented by centrifugation and a lipid-free supernatant be obtained.
Should your lipids carry a negative charge (such as with phospholipids), my colleagues and I always used anion exchange chromatography to remove even traces of lipids from protein preparations. As the charge-per-surface ratio is way higher for small lipid molecules than for proteins and the negative charge is maintained even in acidic pH, buffer conditions can be chosen to bind only the phospholipids and let the protein pass through the column. You should just take care to establish a positively charged protein surface by choosing a buffer pH well below your protein pI (usually about 1.5 pH units).
Hope one of these suggestions works for you. Good luck!
Best,
Joscha
Hi Arlind, most lipases remove the head groups of lipids and not the lipids themselves. If your protein in a phosphatydyl inositol linked membrane protein then it can be cleaved off.
Otherwise you will still be left with proteins embedded in lipids.
One easy way to separate membrane proteins from lipids is by using Triton X-114 phase separation. There are many online protocols for TX-114.
Looking at the protein itself. It can have a net charge at a particular pH of a suitable buffer. This property can be exploited for separation by ion exchange chromatography.
Thank you all for your answers, I realize now I should have added a little more detail to avoid certain answers that would not help in this scenario. Alas, Joscha gave me a good idea as to how I could make this work which I will try.
The lipoproteins were already in TX-114, I simply needed to purify them further out of the solution and simple precipitation would still leave the lipids in solution. Furthermore, the proteins themselves are uncharacterized/unsequenced, and there is a variety of them there that may have different net charges at different conditions, therefore I cannot utilize IEC.
Either way, thank you all again, I think I have found a way to proceed.
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