Total Protein: 6His+MBP+TEV+tagret Protein: pI=6.49, MW=165.315 kDa,
Cleaved Protein: target Protein: pI=8.42, MW=121.625 kDa
dialysis buffer: 20 mM HEPES pH 7.5, 50 mM NaCl, 10% glycerol. (similar to storage buffer in a published paper, I also tried 50 mM KCl same issue.)
Tev used: 1:40 by weight.
I got a good amount of protein after Ni column purification. I kept different fractions of it and I wanted to cleave the highest protein fraction with TEV. So I mix the total protein and TEV and dialyse it. During dialysis most of my protein precipitated. I thought that is for high protein concentration (6mg/ml). So I reduced the concentration of my second fraction to 1mg/ml and had the same issue. I run a gel and saw that most of the cleaved fraction is in the precipitate. I am not sure what to do? How can I make sure that my protein does not precipitate?
Is it some intrinsic thing to the protein that once it is cleaved from MBP it is not soluble any more? In that case what do you suggest?
Also there is a big change in the theoretical pI of the total (6.49) vs cleaved protein (8.42). can that be an issue? What is the solution?
After elution (with imidazole?) from the IMAC column, did you dialyse into dialysis buffer before cleavage or add the TEV directly to the IMAC eluate? If it was the latter, what is the elution buffer?
I did both. Let me explain,
The first time, I added TEV directly to the eluted fraction (50 mM Tris+ 300 mM NaCl+150 mM Imidazole) and then dialysed in to the HEPES buffer as mentioned above.
During this step I had another fraction of elute with a lesser protein content. I put it in a separate dialysis bag without TEV and dialysed in the same HEPES buffer.
The next day I found precipitate in the cleaved dialysis bag but no ppt in the uncleaved fraction.
I thought this might be due to the concentration difference of the proteins,
So, the second time, I added TEV to the already dialysed low protein fraction. and dialysed again in the HEPES buffer. Found precipitation again.
Hope this answers your question.
I'd expect that concentration of imidazole to inhibit the TEV (but, then again, it would begin to act once the imidazole was dialysed away). The only thing I can think of is that your target protein is intrinsically prone to self-aggregation, or that under your expression conditions, the MBP folds correctly and your target protein is improperly folded. The MBP keeps it in solution until it is cleaved away.
Perhaps you could change expression conditions (lowering temperature, use of chaperonins...) to encourage your protein to fold properly. Have you tried expression without a tag?
Hello Sujay,
I think the tag is helping your protein to be in a soluble form. But as soon as the tag is removed, the protein is aggregating. Some protein have high tendency to aggregate at high concentration (1mg/ml is still high concentration for some proteins), while some aggregates due low salt concentration.
I faced similar problem. My protein of interest was aggregating at low NaCl concentration. When I increased NaCl concentration it remained in solution.
So I would suggest you to lower the protein concentration and/or optimize NaCl concentration.
Hope your problem will be solved soon.
Hi
I have experience faced same situation.
At that time I increased glycerol concentration up to 20%
and If MBP is not disturb on your project I would recommend that do exp without TEV treatment
Hi Sujay, firstly I would remind you that many times the use of an MBP tag is to allow the expression of a protein in a soluble form, without the tag ectopic expression can yield insoluble protein products. Therefore MBP attached increases the solubility of your protein of interest. Therefore the buffer you are using is only suitable for the MBP tagged variant of you protein. Rather than dialysis there is a really neat way of carrying out this purification step quicker. That is by after cleavage you run the sample through another IMAC / HisTrap column and collect the flow through.
And here is the most important part, if you find the protein is ppting straight way then you might want to think about altering the buffer as much as you can, different salts is a common trick, ionic strength, level of surfactant, use of small amounts of urea of guanadinium hydrochloride. Some of these may be comparable with TEV to allow cleavage of tags and remaining soluble proteins.
Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques