If your protein is still a mixture of cleaved and uncleaved protein after gel filtration, then I wouldn't have high hopes about a successful crystallisation trial. You generally need a single band on an SDS-PAGE gel after size exclusion chromatography, which is an indication of pure protein. Homogeneous protein might still form aggregates, but you should be able to separate these into their different states by SEC.

I haven't actually used MBP-tags before, but I think they basically behave like His-tags and therefore all you might need is a second purification step after your affinity purification. If this is indeed the case, then your protein will bind to the beads/column via its MBP-tag during the first purification step and you elute it with a maltose gradient. You should then dialyse your protein to remove the maltose (this is necessary for the following steps) and then add your protease to your protein. Incubate your protein/protease mixture as required and then do the second purification step. The second step consists of passing your protein/protease mixture over the beads/column for a second time (this is why you need to dialyse it first, as the maltose would interfere during this step). This time only the uncleaved protein molecules and the cleaved MBP-tags will bind to the column, while your cleaved protein without MBP-tags will go straight through it. Elute the uncleaved protein and the cleaved MBP-tags with maltose and discard them. You can then put the cleaved protein through SEC.

You might need to add a very small amount of maltose to your protein/protease sample before loading it onto the column/beads during the second step and use a very shallow gradient to make sure your cleaved protein comes off the column/beads first while the uncleaved protein and MBP-tags remain bound (cleaved protein sometimes sticks non-specifically to columns/beads). The uncleaved protein and cleaved MBP-tags should come off the column later, once the maltose gradient reaches it's full strength.

Hi there,

Crystallization should be performed from soluble and  homogenous protein solution only. If your sample tends to aggregate spontaneously, it will precipitate in crystallization trials. Crystallization is a slow and controlled precipitation process which allows molecules to get ordered in the precipitate.

I agree that aggregation is going to cause a lot of problems for crystallization - unless your protein is forming a specific aggregate such as a trimer tetramer etc.  In that case you could try to crystalize the multimer but getting conditions right to have only the multimer present would be difficult.

Thanks a lot Antonio for your suggestions.

I would like to inform you that I get a clean band in SDS-PAGE after Size exclusion chromatography. However, my protein elutes in the void volume. The polydispersity index of this protein sample is less than 0.2 but it shows high molecular weight and large radius (upto 40nm) of the particles in DLS. Has anyone faced the similar problem of homogeneous aggregation? Could it be possible that this protein might be forming high molecular weight multimers as suggested by Paul D Jones.

Thank you Dominique Liger, Paul D Jones and Paul Lesbats for your insightful answers.

OK, you meant your protein forms aggregates during gel filtration regardless of whether you use cleaved or uncleaved protein (I thought you meant you were getting a mixture). Sorry for the misunderstanding.

The only case where that has happened to me is with DNA- or RNA-binding proteins that co-purify with their nucleic acid substrates. Some proteins (e.g. viral nucleocapsid proteins) protect nucleic acids from enzymatic attack, so they remain intact even if you add DNAse or RNAse to your lysis buffer.

What works in these cases (and I guess it's worthwhile trying in cases without nucleic acids as well) is to run your gel filtration in a high ionic strength buffer. Use your normal buffer at the pH that's best for your protein (1 to 2 points away from your protein's pI) and add 1 to 2 M NaCl to it (I use 1 M NaCl + 2 M NaBr in 20 mM TRIS pH 7.5 for some of my proteins). The ionic strength of the buffer disrupts ionic bonds between the protein and nucleic acids (and presumably between different protein molecules too) and you get one or sometimes more lower molecular weight peaks during SEC instead of a single massive void peak.

I'm not sure if it's going to work in your case but it's an easy experiment, so give it a try and see what happens.

I developed this method for use during Ni-NTA and SEC in this paper if you want to check it out:

Article Nucleocapsid protein structures from orthobunyaviruses revea...

Thank you Antonio for all of your insightful suggestions. I will surely try high salt concentration during gel filtration.

Many Thanks again.

How about adding cholate or deoxycholate?

Thanks a lot Prof. Seiki Kuramitsu for suggesting the use of Cholate or Deoxycholate. Could you also please tell me the working concentration of it? Thanks