proteolytic degradation of rcombinant protein

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Mon Nov 30 07:55:18 EST 1998


Hi Ricky,

Two possibilities about your problem come to mind:
1. the protein is instable and is degraded inside the bacterium
2. the protein is degraded during purification

If (1) is the problem, try 
a. growing at a lower temperature (e.g. room temperature)
b. adding zinc sulfate to 0.2mM final concentration (inhibits some
bacterial proteases)
c. adding Pefabloc (Boehringer Mannheim), a biocompatible serine
protease inhibitor to your growth medium
d. change strains, BL21-derivatives are often used to decrease
proteolysis. Other E.coli B-strains might also work.

If (2) is the problem, try
a. working in the cold and working fast
b. adding a protease inhibitor cocktail
c. if you use urea for purification of denatured protein, replace by
GuHCl. Urea often causes problems in our hands because obviously
proteases can refold after urea removal and then degrade your protein.
d. of course changing to a strain with less protease activity also helps.

Hope this is helpful,
Frank




Frederik Boernke wrote:
> 
> Hi all,
> 
> I have this protein expressed as a MBP-fusion in E. coli XL-I blue
> using NEB's pMAL vector. Unfortunately I only see a faint band in a
> commassie stained gel. However, western blottig shows me several bands
> all smaller than my fusion protein which to me seems like proteolytic
> degradation. Is there any way to avoid this, like lowering temperature
> or changing host strain.
> Any hints will be appreciated.
> 
> Cheers
> Ricky
> 
> ******************************************************************
> 
> Frederik Boernke
> Research Group of  Molecular Plant Physiology
> Institute for Plant Genetics and Crop Plant Research (IPK)
> Corrensstr. 3
> 06466 Gatersleben
> Tel.  039482 -5 321
> Fax. 039482 -5 515
> http://www.ipk-gatersleben.de



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