subunit separation

EK khatipovNO-SPAM at NO-SPAMuchicago.edu
Mon Nov 25 23:03:27 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 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
Ni-IMAC.

>
> 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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