How to stop protein from preciptating during dialysis - (Feb/07/2006 )
Hi All,
I have looked all over this site for an answer on how to stop protein preciptating during dialysis yet nobody out there has reported their success. I am hoping someone out there can help me!!
I have NiNTA column purified a protein under dentauring conditions (as the protein had formed inclusion bodies and I had to use 8M Urea to solubilse these inclusion bodies as no lower conc would work)
However now I need to cleave off the his tag using a Factor Xa cleavage site, yet the FactorXa activity will be inhibited by the Urea. Therefore it has been recommended that I dialyze the protein into the Factor Xa cleavage Buffer. Seemed like a great idea and worked ok for about 2 hours when the protein started to precipitate!!!, which I have confirmed by coomassie.
So now I have a heap of protein I dont know what to do with as I am pretty sure the FactorXa wont cleave a protein that is insoluble. Do I now have to refold?? But then after refolding can I dialyze into Factor Xa cleavage buffer safely?? I have no idea and very stuck.
Incase you see a way to improve dialysis conditions the buffers are below:
The Purification Buffer contains: 100mM NaH2PO4, 10mM Tris Cl, 8 M Urea pH 4.5
The Factor Xa Cleavage Buffer contains: 100mM NaCl, 50mM Tris HCl, 5mM CaCl2. pH 8
Also this protein is supposed to be used for antigens and rabbit injection ....so I'd heard a rumour that tritonX-100 cannot be dialyzed out therefore cannot be used for antigen preparation..Is this true?
thanks for any suggestions...
Does all the protein precipitate during dialysis? if not you may be able to use what does not precipitate - I rely on precipitation during dialysis for purification of one of my proteins.
If you having problems with refolding look into other refolding techniques - it would seem that this is your problem, after 2 hours sound like when enough of the chaotrope has diffused away that the protein is begining to refold - can't then precipitates.
things to try include: rapid dilution into fast stirred solution
Changing refolding buffer conditions - trial end error you know about your protein - look for simmilar proteins in journals and see how people have handled them. - if factorXa is fussy about conditions then this will not work.
In this case I would suggest recloning into a thrombin cleaving vector, CNBr (cyabogenbromide) cleavage is then an option, the advantage of this is that it is not a protein - but a chemical so does not care about buffer conditions, just that the site is available.
Unfortunately all the protein precipitates.
Sorry but I am new at this protein stuff and I am a little confused about your second line...Do you mean the urea is gone and therefore unfolds and then precipitates? but then I thought that the urea denatures the protein yet solubilises it somehow? Oh no I'm really going in circles now!
quote name='jonboy' date='Feb 8 2006, 11:21 PM' post='40329']
Does all the protein precipitate during dialysis? if not you may be able to use what does not precipitate - I rely on precipitation during dialysis for purification of one of my proteins.
If you having problems with refolding look into other refolding techniques - it would seem that this is your problem, after 2 hours sound like when enough of the chaotrope has diffused away that the protein is begining to refold - can't then precipitates.
things to try include: rapid dilution into fast stirred solution
Changing refolding buffer conditions - trial end error you know about your protein - look for simmilar proteins in journals and see how people have handled them. - if factorXa is fussy about conditions then this will not work.
In this case I would suggest recloning into a thrombin cleaving vector, CNBr (cyabogenbromide) cleavage is then an option, the advantage of this is that it is not a protein - but a chemical so does not care about buffer conditions, just that the site is available.
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well according to what i have read, the rate of aggregation depends on the concentration of the protein unfolded, so, if your protein in 8 M urea is too concentrated, the folding intermediates, which still have the hydrophobic aminoacids exposed, have more chance to interact and aggregate. Why don't you try dilution refolding, with a low content of salt? 150 mM would be ok, at 4° or lower, then dialyse for at least 4 hr. Greetings.
Boquinha
You may lower the protein concentration and gradient dialysis, or desalt by G15