GST Contamination of GST-fusion proteins

Frank O. Fackelmayer Frank at Fackelmayer.de
Thu Jul 26 02:46:22 EST 2001



Eric wrote:
> 
> Does anyone have suggestions on a method of purifying a gst-fusion protein
> from contaminating gst protein without cleaving the fusion protein.  Due to
> gst dimerization, many of the column purification methods I have tried (ion
> exchange, gel filtration) have been unsuccessful.
> 


Hi Eric,
Contaminating GST protein is usually a result of proteolysis. GST itself
is very stable, and if your protein is somehow susceptible to
degradation you will end up with a mixture of your desired fusion
protein and GST as a remnant of degraded fusion protein.
Thus, the best way to "remove" the GST contamination is to avoid its
formation. Try using a different strain (with lower protease activity),
and/or shorter induction times (30min is often better then 3h when
dealing with sensitive proteins). In addition, follow the usual
guidelines to avoid proteolysis during the purification: do all steps in
the cold (starting with a rapid chilling of the bacteria before the are
cracked), add a protease inhibitor cocktail, perform the purification as
fast as possible, etc.

If you are unable to reduce the amount of contaminating GST
significantly, it will indeed be difficult to remove GST later. Affinity
purification with something strongly binding to your protein of interest
(a ligand, or antibody)  may work if you find conditions where this
interaction is stable but the dimerization of GST is abolished.

Frank




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