Despite the vague info - my first guess would be that folding / oligomerization problems are frequently the consequence of "high yield". ( Edited: ) In this case, trying different promoters might solve the issue.

However, if you want the answers to improve from wild to educated guesses, you should be a bit more specific.

HTH

Christian

Actually, it is long story about how that protein is sensitive for concentration and how easily loses its kinase activity. I wish to find somebody working on this, particular protein and that's the reason my question is so vague. However, any ideas about possible reasons of problems with protein dimerization are more than welcomed. Thank you for your comment, for sure I will check that.

Thanks,

Agnieszka

This protein - alas I am not familiar with it and know well, that this is often a prerequisite to give meaningful advise - has been crystalized several times. There is no hint in the literature?

What is the aim of your exercise? Crystallization? Cinetics of the oligomerization? Something else?

How did you check for its oligomerization state?

Surely you can be a bit more specific.

Else, if the oxidation is necessary, you could try to slow down this process, by removing and adding agents through dialysis. Or change the buffer, or add other agents such as trehalose / glycerol. Well, again, there are many, many options. Some will serve. Some will not. Without knowing anything about the construct your are dealing with, the purification steps and conditions, etc..

What I would do with a student of mine (not that I have any, right now ;-) is to brew some tea, take the lab journal and brainstorm with her. Don't you have that option? Alas, mail or forum discussions on such matters are sometimes all too cumbersome.

HTH

Christian

You don't give enough precisions about your problem...

- Are you working with the full-length protein ? If not perhaps you don't have the dimerization motif anymore...

- Does your protein have a Tag ? Because one common issue is that the Tag prevents oligomerization because it is too near from the interaction area. If so, subcloning with the Tag at the opposite extremity of your protein sequence can solve your problem (Cter rather than Nter for example)...

- If your protein is eucaryotic, then there can be problems of folding in E.coli... Expression with baculovirus/insect cells can do the job for this kind of issue...

- You're talking about B-Me and oxidation : have you got any data showing that the dimers form through disulfide bonds ? If so, E.coli strains such as SHuffle or Origami can help forming those bonds...

Hope this will help

Even while it is in principle correct that Tags may interfere with complexformation, this really depends on the type of tag used. GST e.g. is actually promoting dimerization in many cases since it forms homodimers - this can even lead to unwanted dimers in some cases, but if you want to get dimers in the first place, a GST-tag would be worth a try.

In the case of kinase activity of ERK2 however the problem of low activity most probably arises from something different: If expressed in E.coli this protein is almost completely inactive since it requires an activating phosphorylation by MEK1 - something which clearly cannot happen in E.coli. If expressed in eukaryotic cells (e.g. insect cells) it usually can be purified in an intermediatly active form, but fully active ERK2 can only be produced if properly activated by MEK1, for example by incubating it with catalytic amounts of the activating kinase.

cheers,

Daniel

This paper describes the production of ERK2 in bacteria in sufficient quality and quantity for the selection of phosphorylation-state specific binders and for crystallization:

Kummer, L., Parizek, P., Rube, P., Millgramm, B., Prinz, A., Mittl, P. R., Kaufholz, M., Zimmermann, B., Herberg, F. W., and Plückthun, A. (2012). Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries. Proc. Natl. Acad. Sci. U. S. A. 109, E2248-2257.

http://www.bioc.uzh.ch/plueckthun/pdf/APpub0337.pdf

Thank you Annemarie! That is very helpful. I will go through that right away.

Thank you All for all very helpful advices. My problem was solved after going through structure and papers, what you recommended. It seems that dimerization of ERK1/2 is disulfate bonds dependent and breaking down the dimer was an effect of using a reducing agent in my purification prep.

Activity of that protein strongly depends on dimeric state and phosphorylation. In my research I was not able to show full kinase activity of the protein when it was kept in a monomeric form. Seems that it works out now.

Thank you again for all advices I have got from you!

Agnieszka