GST fusion protein

Jongrak Kittiworakarn g3937506 at STUDENT2.MAHIDOL.AC.TH
Wed Jul 8 20:40:44 EST 1998


> Hi Luc:

Did you check your crude E.coli lysate before loading to the glutathione column?You
might found your protien in the crude but not bind to the column as I.

I have an experience in expression of hydrophobic peptide ( 8 kDa and 25 kDa) with GST
fusion.  The expressed protein was in a very high level (30-40% of total crude E.coli
lysate), but it was in insoluble form.   After refolding of the protein, I got the
soluble form.
But the refolded protein neither showed GST activity nor bound to s-hexyl glutathione
agarose.  I think the fusion protein might interfere the GST activity although it have
a propered fold in some extend.

If this is your case, you may try cleaving your protein out with protease. Then purify
the part of your interest by some other method.

Jongrak


> GST fusion protein
>
> Luc CAMOIN (camoin at cochin.inserm.fr)
> 7 Jul 1998 08:33:55 +0100
>
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>   ------------------------------------------------------------------------
>
> To: protein-analysis at net.bio.net
>
> From: Luc CAMOIN <camoin at cochin.inserm.fr>
>
> Subject: GST fusion protein
>
> Date: 7 Jul 1998 08:33:55 +0100
>
> Dear netters,
>
> I expressed a fusion protein produced in E Coli. The fusion protein is a
> GST tagged protein and the predicted molecular weight is around 45 kDa. My
> protein of interest is a very hydrophobic protein of 19 kDa.
> Purification on Glutathione sepharose 4B and western blot with anti GST
> revealed only a band around 25 kDa. This molecular weight and reactivity
> with anti GST could suggest that this protein is GST alone.
> Can we conclude that the contact between GST and the protein of interest is
> a protease sensitive region? Is this a known problem? Did you also find
> this problem in your experiments? Can you give me references about this
> problem?
>
> Thanks in advance,
>
> Luc CAMOIN
>
> Institut _/_/_/_/ _/_/_/_/ _/_/_/_/ _/_/ _/ Luc CAMOIN
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