I am trying to fold a protein which is in urea containing buffer, to its native conformation. I had read that sarcosine is known as protecting osmolytes. Can it be used to aid in protein folding while removing the urea from the solution? What is the mechanism by which it stabilizes the protein?
i have tried arginine, was not of much help. But have not tried cyclodextrin. Can you suggest what concentration should i start with for cyclodextin?
Very well written review with a lot of tricks to apply, I have read as an excellent novel;)
"Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins" by Luis Felipe Vallejo and Ursula Rinas
www.microbialcellfactories.com/content/3/1/11
Good luck - Jan
First I would suggest to try to express your protein using different strains, plasmid, co-expression------. It would be better to do in order to get your protein soluble. Otherwise you can make aconc gradient of beta-cyclodextrin on your own scale. The review is also quit good read it carefully
Structure and effect of sarcosine on water and urea by using molecular dynamics simulations: Implications in protein stabilization
http://www.sciencedirect.com/science/article/pii/S0301462212001470
Did you try TPP (three-phase-partitioning) with t-butanol? it is one of the options for refolding proteins denatured with urea. Maybe it works fine with your protein.
Dilution into deoxycholate followed by dialysis into the final buffer worked for me.
Thanks for thae answeres, I will try the options that all of you have suggested.
Jan Piwowarski- Thanks for the review, it is very informative indeed!
I think sarcosine will do two things: first it will increase the solvation of urea and directly interact with it to reduce urea interaction with your protein. Second it decreases the stability of the unfolded state. You will need very high levels of sarcosine – several Molar – for this to work.
I am not expert in sarcosine. But urea is denaturing agent. Everyone knows it. I have made a lot of experiments and computer simulations of DNA. But I have experience in protein. Sorry, I cannot gove you a piece of advece. George Malenkov
I have reread my message and have found a lot of misprints. I have tried to correct them.
I am not expert in sarcosine. But urea is denaturing agent. Everyone knows it. I have made a lot of experiments and computer simulations with DNA. But I have no experience in proteins. Sorry, I cannot give you a piece of advice. George Malenkov
I have done some experiments and simulations on sarcosine. I think, sarcosine can protect protein from denaturation, however, if protein is already denatured, it may not fold it back. Sarcosine counteract the effect of urea and arginine is known to help protein in refolding, so I would say if you use mixture of sarcosine and arginine in your case, it might be of help.
Narendra
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