Peter Drueckes wrote:
>> Hi everybody,
>> GST or the GST-domains of fusion proteins are known to dimerize. I
> wonder if anybody might know of conditions that avoid this dimerization
> in order to have a defined size of the protein. This would help to
> separate the GST from other proteins by gel filtration e.g. after
> thrombin cleavege of the fusion protein.
>> Alternativly conditions where the dimerization is quantitative could
> also work.
I don't know of any technique for separating the GST dimer
into monomers and can't remember reading anything about it.
As far as I know, the dimer interaction is quite strong and
may be quite hard to separate. You can try and find the
following reference that describe the GST structure:
Lim et al
Protein Sci 1994 Dec;3(12):2233-44
I have not been able to read this paper because my library
didn't subscribe to the journal for the year concern. If
the binding interaction is largely hydrophobic it is
probably not worth trying to separate the dimer. A couple
of things you could try to separate the GST from your
protein:
1) digest on the beads - your protein should come out while
the GST is attached to the resins. Some proteins, however,
do not cleave well on the bead.
2) remove GST after digest by rebinding to GST resins. You
can do that after gel filtration as the glutathione would be
separated from your protein and GST. Works well.
3) use an additional ion-exchange step - this works only if
your protein has a different pI to the GST, but has the
advantage that it can further purify your protein if there
are contaminants. I can't remember which ion-exchange
column you should use (possibly DEAE, but if you are
interested, email me and I can ask someone who has
successfully done it before).
> All suggestions are wellcome.
> Thanks, Peter.
