T. S. Grewal sasgrewl at
Wed Feb 8 05:12:10 EST 1995

  I've been trying to purify a GST-fusion protein (~47 KDa) using
the pGEX-2T system from Pharmacia. I'm having a number of problems
1) solubilising the GST-fusion protein following lysis
  (currently I'm using 1.5% sarkosyl)

2) I'm getting low binding of the GST-fusion protein to
 the glutathione sepharose matrix (~ 50% of total GST
 activity binds)

3) Problems with degradation? of GST-fusion - I get
  quite a lot of GST purified!

4) Cleavage of the GST-fusion protein on the column with
  thrombin. Works OK on a small scale, but not at all well
   when using 10 ml of matrix.

If you can help me on any of these or other points, thanks.
Any body else had any problems/hic-ups with this system?


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