GST Purification

ChenHA Hzhen at freeuk.com
Tue Mar 26 10:47:02 EST 2002


Mark Olson wrote:
> 
> So what kind of buffer would probably break the 50kd GST dimer? I was
> thinking a high salt buffer would prevent the dimerization.

As far as I know, GST is a stable dimer and you can only dissociate the
dimer by drastic means like unfolding (although you can always do a
literature search and I'd be interested to know if I am wrong).  If your
protein in 60 kD, then the total size of the fusion protein is 170 kD,
in which case you might use membrane with a large pore size.  The
problem is that the protein is likely to be separately folded structures
of 2 x 60 kD proteins and one 50 kD dimer, so they might all go through
the membrane.  You can just try different pore sizes and see which one
works.  If you have mixed population of GST dimers with one, two or none
of its fusion partner cleaved, then you might not be able to separate
them cleanly.

The easy way is to use gel filtration which should easily separate the
GST from the fusion protein.  I assume you want to keep the GST portion
of the fusion, but if not, simply digest and then remove all GST by
glutathione sepharose.  

As someone else mentioned, you can try and see if you can avoid
proteolysis.  If not, have a look at the sequence of your protein and
see if you can remove region or mutate sites with are likely to be
digested easily (although this is probably more work than you really
want to do).  You can also check your DNA sequence - sometimes regions
with rare codons in tandem can cause premature termination of the
translation so you only see the GST portion (you can try using codon
plus cells in this case). 

If the GST doesn't affect whatever you want to do, then don't bother
removing it.




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