RE-GST contamination of GST fusion protein
(by grand.david from gmail.com)
Wed Apr 2 03:50:15 EST 2008
Hi Paul.Phelan <http://www.bio.net/biomail/listinfo/methods>
Can you cleave your target from GST with a protease recognition site.
Or, try to use reducing buffer when doing your gel filtration ...
Reducing the GST dimer as monomer will let you separate it from your fusion.
Don't know if for your application reducing buffer will be a limitation. And
I have never tested it.
Do a reducing test, try different DTT or B-mercapto concentrations, load it
on a non reducing SDS-PAGE and find the best lower limit of [DTT or B
mercapto] needed. Probably a strong reduction isn't good for your target
protein so doing this test can be a good information.
Treat your co-purified fraction with this buffer before gel filtration and
see what happen.
You will probably be able to separate them by size, as monomers after
Doing denaturation is good but you will have problems for the renaturation
as it's a difficult process.
If someone can help :)
In <mailman.1253.1206722309.2451.methods from net.bio.net>,
Paul J. Phelan <Paul.Phelan from tufts.edu> wrote:
> To whom it concerns,
> This question maybe have been asked before, but I am having trouble
> with separating GST from a preparation of GST-Importin alpha. I have
> maybe 10-20% GST co-purifying with the fusion protein. The pI of both
> proteins is the same, so ion exchange chromatography does not separate
> them, and even on a Sephacryl S-100 gel filtration column, the two
> proteins (87 kDa fusion, 27 kDa GST) eluted together. Does anyone have
> a clever trick to remove GST from a GST fusion prep.?
> My next attempt is going to be to try gel filtration again but with 0.3
> M NaCl and maybe some detergent to try to separate the proteins.
> Any advice would be greatly appreciated, thanks
GST dimerizes, so it isn't surprising that the cleaved GST moiety
co-purifies with your GST-Rch1 fusion.
In my limited experience with GST fusions, I've never encountered
this premature cleavage that you're seeing, so my suggestion below
is not tested.
Why don't you denature your GST/GST-Rch1 mixture, and then separate
them by gel filtration? Using 3M GdHCl?
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