I am trying to purify an MBP tagged protein from Rosetta-(DE3)-pLysS strain. The gene is cloned into pMAL-c5X vector. The size of MBP is 42 kDa and that of fusion protein is 82kDa. Initially the protein came in the insoluble fraction but tagging with MBP has increased its solubility. The problem that I am facing is in its elution. I am using 100 mM maltose in the elution buffer. The control MBP is eluting well but the problem is with fusion protein. I have tried with buffer containing 200 as well as 500 mM NaCl, but no improvement have been seen. The buffer composition is as followed- 0.5% Triton X 100, 100ug/ml Lysozyme, 50mM Tris pH 7.4, 200/500 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1 mM DTT and 5 % Glycerol. Any explanation why is it happening like this? What should I do to get the protein in the eluted fraction. Thank you.
Is it possible that your protein is precipitating in the column? Also, I would try to lower the NaCl concentration to even 50 or 25 mM with the maltose concentration unchanged for elution. You will get a non specific elution then because of the ionic force level drop but if you removed most contaminants by washing, then you shouldnt have a lot of junk in the elution.
Reasons for this behavior might be unspecific binding to the column - Contrary to common belief, MBP does not really solubilize the protein it is fused to (MBP does not have chaperone activity), it is just the high intrinsic solubility of MBP that (possibly) more or less "drags" its guest-protein into solution. The fusion partner thus might still be misfolded, interacting with column materials - leading to effects like the one you probably observe. Choosing a different fusion tag (with chaperone activity) might help.
But then, some MBP-fusion proteins simply do not elute well with maltose - do you have the possibility to use nickel-affinity instead? Or try to elute with NaCl in ion exchange chromatography?
Try to see whether the protease for removal of tag (TEV, FactorXa etc.) is able to cleave off MBP from the fusion. If its cleaving, try to elute the proteins. Now, if MBP elutes but your protein of interest is still stuck to the beads then you have to look for a different strategy. This will help you to troubleshoot the problem.
After a look at your gel picture I think that you have another problem. If you will see the bead bound fraction, you will notice that there are lot of bands at the molecular weight range spanning the size of MBP and that of MBP-Fusion. This suggests the possibility of ribosome fall off post translation of MBP or degradation of your MBP fusion protein. That is why in the eluted fraction of the experiment done with MBP-Fusion you see bands at the molecular weight of MBP (which might be only MBP as a result of ribosome fall off). However, the higher molecular weight bands do not elute as they have part of your protein of interest which "sticks" non-specifically to beads.
@ Tomasz Janczi- I am also suspecting that my protein is either interacting non-specifically to the bead or it is in the form of aggregates that get stuck into the column. Can you please explain a little on why should I lower the NaCl concentration? I was thinking to increase it further with a hope that it might prevent non-specific interaction of my protein.
@ Peter Rehbein- Yeah I can opt for a different tag e.g., His tag/GST tag. But as I have already started with MBP, it would be good for me if I can elute it with this system. Any suggestion that can be implemented to prevent the non-specific interaction with the bead?
@ Saikat Bhattacharya- I think the protease cleaving experiment can be done to become sure whether my protein is non-specifically interacting with the bead or not. If it does not interact then I can elute it out. But if it does whether increasing the salt concentration further would be a right choice to inhibit the non- specific interaction?
The non-specific bands that I am getting in the eluted fraction might be smaller products that have formed due to ribosome fall off or the MBP alone (as you mentioned)
Thanks to all for your valuable suggestions.
As far as I know, you need ionic strength for any kind of affinity purification. I suggested eluting(only eluting) in a buffer with a very low amount of NaCl to lower the ionic strenth to flush quite everything from your resin. Never did a MBP purification but lots of His tag and with no NaCl the binding was non-existant. Also, you might want to make another round of elution with just the buffer that you are using, but additionally 4M Urea or 6M Guanidine hydrochloride. Of course it would not help you because you would get unfolded proteins but it might indicate that your proteins are precipitating somewhere during the purification.
What you could try if you want to stick with MBP-tag:
You could simply add 10 % glycerol and molar concentrations of arginine to all buffers, even to the binding buffer. This should reduce the non-specific interaction considerably. Worth a try.
Glycerol would reduce nonspecific interaction and arginine could keep the (possibly) misfolded part of the fusion protein solubilized. You would however still have the problem that the protein elutes, but is still misfolded and might precipitate then as soon as you remove the arginine (buffer exchange) or cleave off the MBP.
If I may, I would propose the following to potentially circumvent this:
1. Add arginine to your binding buffer (and probably 1 M urea), this should solubilize the protein enough to prevent direct precipitation on the column.
2. Using a gradient mixer on your FPLC system, do a gradient from 100 % binding buffer to 0 %, slowly removing the arginine/urea let's call this "refolding" or "washing", whatever you prefer. (The "refolding" buffer would be of the same composition as your binding buffer, just lacking arginine/urea.)
3. Elute the protein hopefully properly folded using maltose.
Do not exceed 3 M urea, this will denature your MBP and thus it will not bind to the maltose column anymore.
Best of luck,
Peter
First of all thank you so much for all the valuable suggestions. I tried with different buffer conditions but nothing did work. So I go with a different approach. I did 'on bead cleavage' and got a band of my protein in the gel which I further confirmed by western blotting. As I was doing a small scale experiment, the next step would be increasing the culture volume. Once everything is standardized I would try to get my protein by gel filtration chromatography.
@ Tomasz Janczi- When I started I did not add NaCl to my buffer but later to inhibit the suspected non-specific interaction I used buffers of different salt concentrations (200 mM, 500 mM, 1 M and 2 M). But, the situation was not improved.
@ Grant Shimamoto- Thanks for your suggestions. Yes my protein has several cysteine residues. But I did my experiments under both reduced and non-reduced conditions. I have tried with different concentration of glycerol too. Again so change has been observed. Yeah I can once try with arginine which I didn't.
@ Peter Rehbein- In my case salt, glycerol, buffer pH none of these helped. I could make a try with arginine if my current strategy fails.
Once again thanks for all of your suggestions. It was indeed a learning discussion. Thank you.
Just make sure at an early stage that the eluted protein is really properly folded - otherwise you will waste lots of time with misfolded protein (which might give a positive signal on Western)...
@ Peter Rehbein - If I was Sujay Pal, I would do what you suggest but also do the purification with 1M Urea(also elute in 1M Urea) and then exchange the buffer to no Urea.:)
Because of the cysteine content it is likely that the protein will need to be folded at some point in the process. If done before purification then it might behave well on the affinity chrom. But if after chrom. then it will have to be kept soluble and reduced with chaotropes and maybe TCEP: perhaps at lightly acidic conditions to protonate/stabilize the cysteines followed by a refolding matrix study.
Why not start out with solubilization of the inclusion bodies (IB) rather than the soluble fraction? If refolding has to done regardless of the situation then the IB will have greater starting purity and product quantity than the soluble according to your gels. Going after the soluble material is very tempting but may not fold well due to the greater amount of contaminants in the soluble fraction versus the IB as well as having risk of greater amounts of contaminants that may not clear as expected. Human IgG1 Fc is an example of this.
Hello, currently I am also working with pMAL-c5X vector. I do have a problem during purification. I've tried several methods that were mentioned above, but I still could not get the purified protein. Whenever I check the protein through western blot, I always got 2 bands , which are the fusion protein (around 61KDa) and also the MBP itself. Is it normal for the MBP to appear alone? Thank you in advance .
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