I tried 8M Urea buffer for plant protein extraction and nickel purification. I equilibrated the nickel beads with TEV cleavage buffer which contains 50mM tris, phosphate, 300mM NaCl. Then I added TEV enzyme (1:100) to do TEV cleavage on the nickel beads at 4C overnight. However, I did not get any proteins in the elute the next day. I figured it might because I did not add protease inhibitor during cleavage or 6His-TEV protease attached to nickel beads did not work to cleave off other proteins.
Now I am thinking to do the TEV cleavage after elusion. But I need to remove both 8M urea and imidazole before doing that. Should I
do washing and elusion steps without adding urea, just use phosphate and NaCl buffer. Then desalting the elutes against TEV cleavage buffer to remove imidazole before TEV cleavage? Can I use the same 8M urea buffer for washing and elusion because I need to dialysis anyway? Would proteins elute better under the same condition as extraction and binding? But I saw people mention a series dialysis for urea and I am not sure what is the difference between a series dialysis and one-step dialysis?
Also, some people use TEV cleavage while dialysis, could anyone tell me how to do it?
Thank you!
Let me see if i understood correctly. You had his-tag protease immobilized with the nickel ions and added your sample dissolved in urea? The presence of urea can denature your his-tag protein rendering it innefective. Do you cleavage afterwards as you mention. You can run on-column refolding of your protein. Refolding is always complicated. It is usually run as slow as possible (urea remotion) to promote refolding not aggregation again. This is why people use step dialysis.
TEV protease will not work in 8M urea. The best way to purify your protein is to do TEV cleavage during dialysis. During the wash step in Ni-NTA purification, you can serially dilute the urea concentration to 0M (Going from 8-6-4-2-0M) 5 column volume. Elute the protein in 300 mM imidazole and add TEV protease and put for overnight dialysis. Next day, pass it through fresh Ni-NTA beads, your protein should come out in flow through and TEV will remain bound to beads as it contains his-tag.
Hope it works.
Thank you so much for your answering! Now I know why use series dilution. One question is if I add TEV protease during not after dialysis, should I add it in the beginning of the dialysis or i add until I dialysis against 0M Urea? Thank you!
A follow-up for the TEV cleavage. I was not able to cleave any of my proteins from the 6His tag by either dialysis. I am suspecting that using 8 M urea for extraction might damage the TEV cleavage site fused to my target protein and the damage is irreversible. That may explain even I change from 8 M urea to mild buffer, the TEV enzyme still does not work. I am now trying Guanidine-HCl for protein extraction.
Urea will not damage a cleavage site. You will get the same result with guanidine. Something else is happening.
Thank you for your kind reply. I am guessing TEV enzyme recognizes native proteins better than denatured proteins....and few proteins can refold after using 8M urea for extraction. I am going to try the other extraction buffer. Also, I was not able to find any literature which has the TEV cleavage step after using 8 M urea for protein extraction.
Why do you think the cleavage site cannot be damaged under a strong denaturing condition? Do you know anyone who has done TEV cleavage after denaturing protein with urea?
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