Sigrid Van Boxstael
svboxsta at vub.ac.be
Wed Nov 27 05:51:18 EST 2002
> "Sigrid Van Boxstael" <svboxsta at vub.ac.be> wrote in message
> news:3DE24661.97428347 at vub.ac.be...
> > I am studying a protein which is a dodecamer, composed of two different
> > polypeptitdes one assembling into trimers and the other one into dimers:
> > so the complete protein consists out of two trimers of one polypeptide
> > (the catalytic subunit) and three dimers of the other (the regulatory
> > subunit). At the N-terminus of the catalytic polypeptide there is a
> > His-tag linked. By using a Ni-column, it was possible to purify the
> > protein. Now I am interested in separating the regulatory subunit from
> > the catalytic subunit.
> > I thought the following would be a good idea :
> > 1)Dialysis of the protein (95 % pure) against 8 M urea, 0.1 M tris pH
> > 8.2, 10 mM mercapto-ethanol; Purpose : unfolding of the protein so I
> > get free his-linked catalytic polypeptides and free regulatory
> > polypeptides.
> > This dialysis happenend during two hours at room temperature.
> > 2) Putting the dialysed protein on the Ni-resin. Purpose : The
> > catalytic polypeptides stays on the resin, the regulatory will be in the
> > supernatans.
> > 3) Putting the supernatans on SDS-gel to check.
> > What do I see on the SDS-gel with the supernatans : Nothing.
> > (The gel and staining were OK, the dialyzed protein shows both bands)
> What did the fraction you eluted from the Ni-column show? Or you did not
> elute it hoping to just purify the non-6his subunit?
I did it on small scale in an eppendorf with 1 ml Ni-resin.
It was intention that all the his-tagged catalytic subunits would attach to the
Ni-resin and the regulatory would be free in the
> I would try eluting the proteins step-wise by increasing concentration of
> imidazole to see if the regulatory subunit can be eluted before the
> catalytic one. There is also a possibility of non-specific binding of your
> proteins to the Ni-resin. I am not aware whether 8M urea makes proteins more
> prone to sticking to chromatographic matrices, but for a change I would try
> guanidinum-HCl or guanidinum-sulphate to denature the protein prior to
I think it is a good option to try GuHCl or GuSO4 and as DK suggested heating a
little bit, because my protein
is a hyperthermophilic one and feels very happy at 96 °C.
I thought the big bottle neck would be the "reversible"refolding, but it appears
to be the unfolding (if the regulatory does not stick in a non-specific
way to the column). I will now increase the imidazol concentration in the resin
(the concentration at which it normally elutes) and see either I can detect both
subunits in the supernatans.
Loading a little bit of the resin (before I increased the imidazol concentration
and boiling in SDS-loadingbuffer) on SDS-gel would also tell where the protein
Is there a simple way to see either a protein is unfolded ?.
I know it is possible by Circular Dichroism, do you think this is the best way
to test this instead of trying "blind" things?
Thank you all for your suggestions.
> > As possibilites I see
> > 1) The protein did not unfold and everything is attached at the Ni-resin
> > 2) The regulatory precipitated in the presence of urea?
> > Does anybody has any comments or suggestions to solve this problem or
> > maybey a much easier way to separate both subunits?
> > Can a protein precipitate in such a huge amount of urea?
> > Thanks in advance,
> > Sigrid
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