Hi there,

As the histag is present (positive detection in WB) then it is a matter of tag accessibility for interaction with the matrix. To get sure of this, you should try the purification in the presence of different concentrations of a chaotropic agent (urea or GdmCl). Low concentration of those are not denaturing but allow exposure of normally hidden part of the protein. If the tag is completely buried into the protein structure then denaturing concentration should be required to observe an interaction with the matrix.

Thank you so much! I was also thinking of denaturating the protein to understand whether accessibility is problem or not. But it is an enzyme and I need the activity of it. So it would be a temporary solution for me. That's why I was hopeless but as you said lower concentrations of GuanidineHCl (I assume)and urea is safe I can try it. Do you know what is the safe concentration of urea or Guanidine HCl that preserves the activity as well as open the tag region. It must depend on the protein of interest but anyways..

And If you know can you explain how it opens the region without affecting activity ? Or do you mean activity may be lost although it doesnt get denaturated completely ?

Thank you so much again.

Dear Sinem,

I agree with Dominique regarding the cause of your trouble: obviously, the His tag is there, but not accessible. As he suggests, you may try a mild denaturation and test various concentrations of chaotropic agent in order to make it more accessible and then remove the chaotrope by dialysis. However, if it turns out to be impossible to unmask the tag without denaturing the enzyme irreversibly, you may also try to improve the accessibility of the tag by switching its position to the NH2 terminus and/or by adding a linker between it and the rest of the protein

Dear Sinem

i think that there are 2 possibilities:

1) The tag is unstable and degraded during the bacterial growth.

2) The tag is not well exposed because during the protein sintesys it is folded inside the chain.

i think that the approach reccomended by dominique (imac purification with denaturant) or alternativelly a WB with anti-his is usefull to assess the first point.

Because if the his is it lost, you can try also to change expression condiction (eg lower temperature o shorter induction time to see if you solve it) while in the second case you need to re-desing the clone (e.g use the N-term his or change the link sequences between the his and the protien) and see if in this way you acheive bettere exposition.

good luck

Manuele

Dear Pierre and Manuele,

Thank you so much. These are indeed quite useful. I will try your advices.

You asked for concentrations of guanidine or urea to try, without completely unfolding the enzyme.

Guanidine's effect is usually a bit stronger than for urea at the same molar concentration. I would definitely not go beyond 3M for either one. I would try the effect of 1M; if this does not help, try 2M, finally 3M.

Be aware that while both guanidine and urea are chaotropes, they work a bit different. Guanidine, as it is charged, will mainly work on charged and polar regions of your protein; urea, as a non-charged molecule, will mainly act on hydrophobic and uncharged regions and residues.

Thank you so much!

Hey

Just check in pymol weather your C-terminal his tag is outside or lying inside the core of the protein. If theC-terminal his tag is going inside of the protein, then you need to keep His Tag at the N-terminal of the protein (if N-terminal end is on the surface of the protein). Pymol gives strong information about the accesibility of the N- or C-terminal ends of the protein to the Ni-NTA or Co-NTA affinity columns.

All the best.

Manoj Thakur Thank you so much. I didn't know that one. Do you know If pymol also shows the most probable structure when tag is added ?

Dear Sinem Ulusan,

Do Not add the tag to the sequence you are using in the pymol. Just see the position of the N-terminal and C-terminal end. weather they are inside or outside. If your protein of interest is folding in such a way that both the ends are inside, then you will find very less affinity of those recombinant proteins to the Ni-Nta columns. But it will definitely give you a hint that you have to keep N-terminal his tag or C-terminal His tag in your protein. This strategy has worked for me. I have removed the tag from C-terminal to N-terminal. This lead not only to the increase in the binding capacity of the protein to the column but also increased the specific activity of the protein.

Manoj Thakur

Yeah that one I have checked. I am also doing the same strategy for my own protein now hoping same outcome. I just wondered if I can see the tagged version as well.

Thank you so much.

Dear Sinem,

Did you able to solve the problem or find out any possible reason especially with pymol. I am interested to know as I have previously encountered this common problem.

Thank you .

Sinem Ulusan i think small tags like 6XHis or 10X His should not create any problem in pymol. May be bigger tags like GST can lead to folding issues (complete 3D structure) in the pymol. You can try

best

Manoj

I may be a little bit late here: But did you buffer contain EDTA or DTT in some form?

Sebastian Lühn,

Thank you but no, there was no EDTA or EGTA or DTT..

It turns out that the tag is not available enough for the Ni-NTA due to its conformation...

If after 5 months of trying you still have problems with the IMAC, I would forget about it and try normal protein purification, especially, as you are working with an enzyme. The tag cab easily change its behavior.

As a first step , II would try ion exchange chromatography with a gradient elution step first.

What is the pI of your protein?