I'm trying to obtain a good preparation of my protein for crystallization analysis, but after the first step of purification (his-tag) I always have protein stability problem.
Daniela, can you provide some more details about the protein that you are working with? That will help others to understand your problem in a better way. I hope these suggestions will be help to you:
1. Adding protease inhibitor cocktail tablets (http://www.roche-applied-science.com/shop/products/complete-ultra-tablets-edta-free-glass-vials#tab-0) surely protects your protein from degradation. Likewise, PMSF also helps, but not always.
2. You can perform a thermal stability assay such as differential scanning fluorimetry or a CD assay and screen for various components that could stabilize your protein.
3. Are you working with an enzyme that is a protease or behaves like a protease i.e. self / auto-cleaving? If so, you can try to mutate the potential residues that could be catalytically active and thereby prevent self-cleavage.
Daniela, can you provide some more details about the protein that you are working with? That will help others to understand your problem in a better way. I hope these suggestions will be help to you:
1. Adding protease inhibitor cocktail tablets (http://www.roche-applied-science.com/shop/products/complete-ultra-tablets-edta-free-glass-vials#tab-0) surely protects your protein from degradation. Likewise, PMSF also helps, but not always.
2. You can perform a thermal stability assay such as differential scanning fluorimetry or a CD assay and screen for various components that could stabilize your protein.
3. Are you working with an enzyme that is a protease or behaves like a protease i.e. self / auto-cleaving? If so, you can try to mutate the potential residues that could be catalytically active and thereby prevent self-cleavage.
Ad protease inhibitors to your buffers. These are available from a lot of the usual supply companies as ready-to-use tablets or solutions.
Do you have any protease inhibitors in the buffers? DO NOT USE EDTA AS AN INHIBITOR, it will block Ni/Co binding.
Sigma P8849-5ML Without EDTA - Use at 5x suggested concentration.
Or
Pierce 78425 Also at 5x concentration.
This should be in all buffers including column buffers until you know degradation is stopped at a particular step.
Also, it depends also on how the gel looks. Is there several degradation products or less?
Is your protein in inclusion bodies?
Some more info, if you can?
I agree with everyone. More info is needed. And if itself is a proteinase, then purifying in the presence of an inhibitor or mutating a catalytic residue may be necessary as others have stated.
If it is not a proteinase, but is an enzyme, add its substrate in the buffers. You can get rid of it later. The substrates and or inhibitors usually keep the enzyme in a closed conformation. The same is true if it is some kind of receptor binding a ligand.
Thanks to everyone for the advices...I alway add in all my buffer (except for gel filtration buffer) the protease inhibitors...I express in BL21 the pETDUET vector that contains 2 my protein....one of these is tagged with 6xHIS...(I need to obtain a really pure complexes to try to do some crystalographic experiments, and for these reason I need to perform tree steps of purification 1-his column;2-ionic exchange;3-gel filtration.
My "first" protein of the complex is tagged with his, the second that interacts with the first is untagged. After his-purification, i noted only 1 band (apart the bands of right sizes of my 2 protein), that could be a degradeted product...but, after TEV incubation (4h at 4°C in agitation) i always see many degradation products. Can anyone suggest me how can i solve this problem, i need to obtain the full lenght of my second protein, because for me was simple to have only the domain of interaction (with the other one) but for further studies I need to obtain a full lenght...Thanks in advance
Daniela
TEV cleavage gives many products until you purify everything away again with the Ni-NTA resin. There is usually TEV itself, and the piece that is cleaved off, your mature protein, and perhaps some uncleaved product. So after cleavage, since the His tag is usually on the piece that gets cleaved off, another Ni NTa purification needs to be done and then ion exchange and or gel filtration. If the protein fragments you see come from your own protein which you can determine by Mass spec, then you may need to try to stabilize the protein. This can be done by additives.
You may want to purify the proteins first, without the TEV cleavage, and save that for your last step, and then use Ni NTA to clean it up.
Should be an option, but in the last step of purification after TEV cleavage in the flowthrough of column I'll have my protein without his-tag, but also the TEV enzyme....
TEV is recognizing an really unique sequence ENLYFQ(G/S). Thus I was always adding it in excess, with no worry about the digestion outside the sequence. This way I was successful with purification of many yeast protein complexes. Suggest to find another suspected;)
Add AEBSF 10mM in all your buffers..also do remember these protease inhibitors have a half life and you be aware that you cant rely on them for long...if stability is a problem, protease inhibitors followed by speed is the key to success. you should finish, affinity, ion exchange and gel filtration in about 16 h all at 4C
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