Sigrid Van Boxstael
svboxsta at vub.ac.be
Wed Nov 27 05:30:52 EST 2002
Artem Evdokimov wrote:
> "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:
> Aspartate Carbamoyltransferase by any chance ?
Very good guess! It is the ATCase from the hyperthermophilic archaeon
> > 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.
> Wouldn't it be easier to express these subunits separately ? Have you
> already tried that ?
We expressed the catalytic subunit separately and this worked fine as the
expression is reasonable and the protein can be easily detected by measuring
the activity. For recombinant expression in E. coli we tried several things
also taking to account the special codon usage. The regulatory subunit is
another story, as the expression is
very low (we always have a huge amount of free catalytic subunit left) and
there is no easy way to detect it.
> > 1) The protein did not unfold and everything is attached at the Ni-resin
> Maybe not completely unfold. Similar cases have been reported.
> > Does anybody has any comments or suggestions to solve this problem or
> > maybey a much easier way to separate both subunits?
> I would still try separate expression. Also, if you're expressing these
> proteins from separate plasmids you can try to adjust the levels of
> expression so that there's more of one than of the other. Then your
> preparation would be subtractive, provided that you can purify the non-his
> tagged monomer via conventional methods.
I don't give up yet on the separation of the subunits, but there might be
finally no other option than working on separtate expression.
Thank you for your suggestion.
> > Can a protein precipitate in such a huge amount of urea?
> That's very rare, but I guess not 100% impossible...
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