Removal of GST from digested fusion protein

krishn pratap singh via methods%40net.bio.net (by kpsinghniper from gmail.com)
Mon Jan 16 04:05:34 EST 2012


May be GST interacts with your protein. Is your protein having
Cysteine residues (specially in the active site). What is the function and size
of your protein. I was facing the same problem.


On 1/14/12, AllisonH <nospam from gmail.com> wrote:
> On 13/01/2012 12:29 PM, Phelan, Paul J. wrote:
>> I am trying to purify a protein, starting with a fusion that is
>> double-tagged with GST and poly-His, and my protein of interest also has
>> an n-terminal poly-His tag of its own.  So the fusion protein is
>> polyHis-GST-polyHis(Protein).  An initial step with glutathione Sepharose
>> works just fine, the fusion protein binds and is eluted with 10 mM
>> glutathione.  Digestion of the fusion protein (with His-tagged TEV
>> protease) also works, and gives me the polyHis-GST tag, plus
>> polyHis(Protein).  BUT - when I re-run the digested protein over
>> glutathione Sepharose, the polyHis-GST is not removed, and just passes
>> through the column, along with the polyHis(Protein).  Has anyone else seen
>> a problem with GST from a digested fusion protein not binding to
>> glutathione Sepharose?  Could it be that the GST still has glutathione
>> tightly bound to it?  (The fusion protein is digested by extensive
>> dialysis with TEV protease).  Could it be that the polyHis-GST is not
>> behaving the same as non-tagged
> GST?  This seems doubtful, since the fusion protein binds to glutathione
> Sepharose in the first step.  Before anyone suggests using a Nickel resin to
> remove the polyHis-GST, that's also a problem, since my protein of interest
> (and also the TEV protease) is His-tagged too.
>>
>> Any suggestions/insights would be welcome!
>>
>> Paul Phelan
>> Tufts University
>> Boston
>>
>
> If you have a protein that already has an internal His site you should
> be able to purify it on a nickel column without any tags. In fact, by
> adding a second His tag you probably will not be able to elute it
> easily. (I had exactly this problem a few years back).
> Allison
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-- 
With regards,
Krishn Pratap Singh
Dept of Biochemistry
R.M.R.I.M.S. (I.C.M.R.)
Agamkuan,
Patna-800004
Phone no:  09304763794



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