subunit separation

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Mon Nov 25 19:06:48 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:

Aspartate Carbamoyltransferase  by any chance ?

> 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 ?

> 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.

> Can a protein precipitate in such a huge amount of urea?

That's very rare, but I guess not 100% impossible...

A.G.E.





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