Please help me! I am really tired of this stupid protein purification! - (Nov/13/2007 )
Dear All,
Recently, i am struggling with one unstable protein purification.
My protein is a fusion protein mutant. The corresponding wild type is stable and it is easy to purify. The mutant protein has a His-tag and is expressed in E.coli. The expression is usually very high, but when i lyse cells, 90% of the mutant protein is in the pellet and 10% is in the supernatant where most of the fusion protein is clipped into two proteins. What's more, the rest of mutant protein continue to precipitate and be clipped during the next steps of purification.
I usually start with 10 L of culture. After the last step of purification, I can only get 1 mg if i am lucky.
From this useful forum, I learned that lowering the induction temperature (18 degrees for 16hr) might increase the stability of the protein. But this does not work to the protein i am working on (I tried).
Could anybody help me out! Any suggestion will be greatly appreciated!
Thanks a million in advance!
Recently, i am struggling with one unstable protein purification.
My protein is a fusion protein mutant. The corresponding wild type is stable and it is easy to purify. The mutant protein has a His-tag and is expressed in E.coli. The expression is usually very high, but when i lyse cells, 90% of the mutant protein is in the pellet and 10% is in the supernatant where most of the fusion protein is clipped into two proteins. What's more, the rest of mutant protein continue to precipitate and be clipped during the next steps of purification.
I usually start with 10 L of culture. After the last step of purification, I can only get 1 mg if i am lucky.
From this useful forum, I learned that lowering the induction temperature (18 degrees for 16hr) might increase the stability of the protein. But this does not work to the protein i am working on (I tried).
Could anybody help me out! Any suggestion will be greatly appreciated!
Thanks a million in advance!
What protease inhibitors do you use in your preps? Perhaps you can purify from the pellet with a strong cocktail of inhibitors to stop/reduce the clipping. Does purification at low temp help?
Hi, Swanny, thanks a ton for your reply! The inhibitors i am using are 1mM PMSF, 0.1mM TLCK, 0.01mg/ml leupeptin and 0.01mg/m trypsin inhibitor. i purify this protein at 4 degrees for all steps. I have never worked on pellet with strong inhibitor cocktails before. a stupid question to ask, Could you tell me how to resuspend pellet and make the protein soluble since there are so many debris? Thanks a lot!
plzzzzzzzzzzzzzzzzzzzzzzzzzzz! more help will be greatly appreciated!
besides lowering the temperature of induction you can also reduce the inducer (iptg?) to try to prevent your protein from being in the inclusion bodies.
you can solubilize the pellet in 8M urea, purify the his-tagged protein (still in urea) then dialyze the urea out to try to refold the (purified) protein.
Recently, i am struggling with one unstable protein purification.
My protein is a fusion protein mutant. The corresponding wild type is stable and it is easy to purify. The mutant protein has a His-tag and is expressed in E.coli. The expression is usually very high, but when i lyse cells, 90% of the mutant protein is in the pellet and 10% is in the supernatant where most of the fusion protein is clipped into two proteins. What's more, the rest of mutant protein continue to precipitate and be clipped during the next steps of purification.
I usually start with 10 L of culture. After the last step of purification, I can only get 1 mg if i am lucky.
From this useful forum, I learned that lowering the induction temperature (18 degrees for 16hr) might increase the stability of the protein. But this does not work to the protein i am working on (I tried).
Could anybody help me out! Any suggestion will be greatly appreciated!
Thanks a million in advance!
Depending on what you're using it for, you may not need to have native protein. Preparing the protein in denaturing buffer, using 8M urea might be easier and if necessary you can then try to refold the protein. Another thing that might work is to move the HIS tag to the other end of the protein.
Hi yuer,
How are you lysing the cells? I was using lysozyme and sonication but was getting a lot of aggregation and degradation of my protein so switched to lysozyme only, followed by addition of 0.2% Triton X-100 and 1mM DTT to further reduce aggregation. My protein is now around 70% soluble and purifies nicely.
Going on from mdfenko (the elder)'s comments, I was wondering about possible codon bias for the mutant. Have you checked for a bottleneck at the point of mutation? If you have a low-frequency codon, your expression will slow down which could lead to the inclusion body problem.
Thank you all for your replies! I really appreciate your useful suggestions!
I have two aims to purify this mutant protein. One purpose is to test if this mutant protein is unfolded, the other one is to see the its degradation to compare with wild type degradation.
So if i use urea, i am afraid that it can not refold properly. Still, I will try to use this method.
To Penguin, I am using lysozyme and sonication to lyse cells. It is really a good idea not to use sonication. I always have problems with sonication.
I will try to add triton next time when i lyse them. Maybe i can not add DTT since I have to purify it using Ni column for the first step. Is there other reagent i can add instead of DTT? One more question, if you don't sonicate, how do you know cells are lysed well?
Thanks a million! Everybody, have a good weekend!