subunit separation

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Thu Nov 28 00:29:04 EST 2002


> Very good guess! It is the ATCase from the hyperthermophilic archaeon
> Pyrococcus abyssi.

Which probably has something to do with the fact that it does not unfold in
urea ! We have had hyperthermophilic proteins that *only* unfolded in 6 M
guanidinium isothiocyanate at 50 C ! They laughed ad Gu*HCl and urea.

> 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.

> 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.

Try very harsh unfolding. Also, as DK has mentioned - preparative gel
purification may prove useful, depending what do you need the protein for.

Good luck

A.G.E.





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