Cleaving GST-fusion proteins
aw at dna.bio.warwick.ac.uk
aw at dna.bio.warwick.ac.uk
Fri Oct 6 11:19:23 EST 1995
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
UK
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