Dear Ahmadullah.

Is your protein a putative metal binding protein or it contain many cisteines?

Any proteins is different and to provide rational suggestion i need to konw more details about it, but i suspect that you can have two possible issue:

1) Your protein strip some metal from His-tag coloumn and the metal promote the protein aggregation. Add some EDTA to the eluted fraction and check is the protein is still unstable.

2) Do you have free cisteines that with the time induce protein aggregation via S-S bonds.

You can check it by rresuspend you precipitate protein and load it in reducing and non reducing SDS page. If you have intramolecular S-S bonds, you will observe HIsgh MW aggregates in not recuding confition.

In this case you can:

- add reducing agent (ag. 1mM TCEP) in the buffers and see if the sample become more stable.

- Try to change the expression conditions (eg periplasmic expression, origami strain) to promote the intramolecular formaton of "good" S-S bonds if those need to be present in your protein.

Good luck

Manuele

Dear Ahmadullah,

In agreement with Manuele, you could try to change expression conditions (for example Arctic expression) or add a solubilization tag (e.g. GST) to your protein, which you can cleave after the purification (in case you did not do it already).

Another method would involve to fully unfold your protein and refold it afterwards again. Hereby you can use chaotropic agents (Guanidine or Urea), in doing so you might achieve high solubility. From your post, I understand, you want to crystallize the protein. In this case, my second recommendation (use of chaotropic buffers) might be counterproductive.

You could also use another purification methods, like introduction of chitin binding domain (CBD) or as I wrote earlier GST- or MBP based purification.

I hope this helps

Best regards,

Can

thanks manuele & can

1- there are 7 cysteine residue present in my protein.

2- Immediately after elution, I have dialysed protein against buffer containing 2mM EDTA. still protein got precipitated in dialysis tube.

3- I have also cloned protein under GST tag, but not yet grown the mass culture of that.

Concerning NaBr. I would test salts from different alkalic metals and halogens. NaF, KF, KI ...

I have very good experience with addition of amino acids for stabilization.

Mostly 50mM Arg + 50mM Glu. I do not have time to find citation. But I would also try histidine. The imidazol ring is a part of this amino acid.

Dear ahmadullah,

i noticed that the salt concentration is lower in your elution buffer than in your lysis buffer. Is this intentional? The first thing to try might be just to keep that the same.

My second piece of advice is to use a different metal for IMAC. Some proteins are precipitated by Ni, and I’ve seen this be irreversible even using lots of EDTA. If I were you I’d try using zinc instead of Ni. You can use the same column. Strip it as you usually would with EDTA and then just recharge with zinc instead of nickel.

cheers,

georg

I think the sudden reduction in salt concentration(for crystallization) would cause precipitation in protein, that's why, I reduce salt concentration in the first step at elution stage and further reduction of salt done during dialysis or SEC.

The use of Zn instead of Ni, would not affect the binding of His-tag protein to the bead?@Georg

Zn will still bind his tags. You can even buy this resin commercially. It won't bind them as stringently, so be careful when you load the protein, but that's the whole point here. If your protein is crashed out by Ni, you want to use an alternative metal for which it may have a lower affinity. It's worked for us before.

It's also worth ruling out the salt concentration as a problem. If you protein becomes in soluble in lower salt concentrations, you can use this to crystallize it.

A point to consider: If your protein comes from the cytosol, it is used to a high K, Mg and low Na, Ca environment, which is reducing. For proteins from the interstitium, or from organelles that are topologically outside (e.g., ER), it is the other way round, and the environment is oxydising. Also note that Tris has a K-like, imidazole a (weaker) Na-like effect.

Temperature may also play a role, some proteins are insoluble in the cold.

Dear Ahmadullah,

it has already been mentioned, but I would recommend to elute with Histidin instead of Imidazol. I had good experiences with that.

Also if you coelute the Ions from the IMAC column you can try using "Talon resin". We had a protein that would always "steal" the ions from the chelating columns but it worked with Talon resin.

Good Luck!