GST contamination of GST fusion protein

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Sat Mar 29 07:14:06 EST 2008


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?

AC
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