Cleaving GST-fusion proteins

[randal berg]

In article <453kub$b7p at violet.csv.warwick.ac.uk>, aw at dna.bio.warwick.ac.uk
wrote:

> 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

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.

Good luck

-- 
randal berg                  
Department of Molecular Medicine  
The University of Auckland         
Auckland, New Zealand

E-mail r.berg at auckland.ac.nz