Cleaving GST-fusion proteins
r.berg at auckland.ac.nz
Mon Oct 9 17:08:08 EST 1995
In article <453kub$b7p at violet.csv.warwick.ac.uk>, aw at dna.bio.warwick.ac.uk
> I have constructed a fusion protein using the pGEX-2T vector
> (Pharamcia). The protein of interset is expressed (confirmed
> by Western blotting) and I am able to purify it to a high degree
> using Glutathione-Sepharose 4B.
> Now for the problem.........I want to cleave my protein from
> GST, using Thrombin. I have attempted this but the protein
> of interest and the fusion protein appear to be
> eluted at the same time.
> Therefore, for some reason the cleaved glutathione-S-transferase
> is not binding to the column and I am ending up with two bands on
> the SDS-PAGE gel.
> Can anyone help me to obtain a purified cleaved protein?
> Thanks for your help.
> John Lloyd
> University of Warwick
I have been using TPCK-treated trypsin at 2 to 5 ug/mL (1 hour at 37C) to
cleave a variety of GST-fusion proteins. The trypsin cuts at the same
site as thrombin, and as long as your protein is not sensitive to trypsin,
this might work for you.
Department of Molecular Medicine
The University of Auckland
Auckland, New Zealand
E-mail r.berg at auckland.ac.nz
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