I expressed a recombinant protein in a procariotic system but I need to eliminate endotoxins, LPS, I tried using polyLysin resin but it doesn't work (I loose too much protein). I read that using polymixing B could be better and also to try bacteria producing less LPS or modified (Clear coli).
Does anyone try any of those methods. I would appreciate any suggestion.
Try the advice here, it's excellent: http://www.proteinchemist.com/tutorial/endotoxin.html
Hope it helps!
Thanks for your answer. My recombinant protein comes from GST-fusion and it is in TrisHCl 50 mM, NaCl 150 mM, DTT 1mM pH 7.0, so I need to begin with the cation exchange and follow with the anion exchange?.
We used to have good luck using the polymyxin B resin. Another suggestion is to consider expressing the protein in the baculovirus/insect cell expression system.
The ClearColi work very well in my experience and you don't need to change your purification protocols.
When using normal E.coli, you can also somewhat reduce the LPS content by adding Triton-X 114 to your wash buffers.
Thanks all the answers, but I have another question: I need to use my protein in cell culture and animals, so using detergents as Triton I will need further to eliminate them? and how I can do it.
you can eliminate everything that's smaller than your protein by dialysis against a buffer of your choice.
Thanks for the suggestions,
Another thing: what brand of commercial poymixin B should be better?
Dear Thomas, that is not fully correct. Some detergents are not dialysable. Once they stuck to protein they stay there tightly bound (SDS). Others form micelles too big to be dialysed (Triton). http://wolfson.huji.ac.il/purification/PDF/detergents/PIERCE_DetergentRemoval.pdf. In our hands NiNTA in presence of 0.2% NP-40 in first buffer, then TEV cleavage, then 2nd NiNTA and gelfiltration results in preparations
good point, didn't take the micelle formation into account. thanks for the comment
Have a look at this paper, its a very simple and effective method to reduce endotoxin levels in rec protein preparations.
http://www.sciencedirect.com/science/article/pii/S0009912097000490
Good luck.
If you use cytoplasmic expression you may consider to do spheroplasting prior cell lysis. EDTA strips most of the LPS of the cell surface. This way there will be lower LPS content in cell lysate to begin with. If your protein/purification protocol can tolerate acidic pH this would also decrease LPS contamination. Polymixin B resin is very efficient in LPS removal.
Researchers have had good success cleaning up E.coli expressed proteins using EndoTrap HD - resin coated with phage protein having a high affinity to LPS. You can find more information on Endotrap below.
https://ilexlife.com/products/endotrap-hd-endotoxin-removal-products
Anna Konovalova Thanks for your answer. I am aware of the use of low pH to get rid of LPS. Do you know the mechanism of how this works? does LPS washes off at low pH or it remains bound while protein is eluted? any reference you may have? Thanks
Nazish Abdullah LPS becomes protonated when pH is low, loses its bound cations, and disperses easily in solution, hence can be easily washed off.