Activity restored by mixing recombinant domains?
roney.graf at uni-konstanz.de
Tue Oct 28 04:42:48 EST 1997
In article <fof1-2810970941380001 at zellkultur.biologie.uni-konstanz.de>,
fof1 at chclu.chemie.uni-konstanz.de (Frank O. Fackelmayer) wrote:
> In article <lloyd.graham-2908560305100001 at tastiger.dbe.csiro.au>,
> lloyd.graham at molsci.csiro.au (Lloyd Graham) wrote:
> > Greetings all,
> > I'm looking for any precedents where the domains of a monofunctional
> > enzyme have been expressed separately as inactive proteins, but when mixed
> > together they associate to form a complex with significant enzymatic
> > activity.
> > The closest I have got to an example is mouse creatine kinase, where
> > independent expression of the two domains gives inclusion bodies but
> > co-expression yields active soluble enzyme [Gross et al., 1996: Prot. Sci.
> > 5, 320-330]
> > If anybody can provide instances where purified inactive domains
> > complement in vitro, I would be very grateful.
> Hi Lloyd,
> What about alpha-complementation of beta-galactosidase, used by everyone
> as the "blue-white" selection in cloning procedures. In this case, a short
> N-terminal part (the so-called alpha-peptide) is encoded on a plasmid, and
> the remainder of the protein is encoded by the E.coli host genome. Neither
> of the two parts alone is enzymatically active. Galactosidase activity
> (blue staining of the colonies in presence of IPTG and X-gal) is only
> present when both parts of the protein are present, i.e. no insert has
> been cloned into the alpha-peptide encoding vector that would disrupt the
> coding information.
Another example would be aspartate transcarbamoylase from E. coli. The
catalytic subunit has been expressed in two fragments which don't even
correspond to the two domains of the protein - you can't really do that
without a major rearrangement of the polypeptide. As in your own example,
the fragments expressed individually are insoluble and are purified as
inclusion bodies, but co-expressed they associate. However, you can
denature the individual fragments with urea, mix, and renature, and you get
the activity back.
check Yang, Y. R. and Schachman, H. K., Protein Science 2/1013-1023/1993
More information about the Methods