Proteases in E. coli strains

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Sat Nov 15 20:48:48 EST 1997


In article <64kdi1$dt8 at gazette.bcm.tmc.edu>, simonh at bcm.tmc.edu (Simon
Hoffenberg) wrote:

> Hi netters,
> 
> We have a problem expressing one of our proteins. Unlike more common
> problem when a protein is insoluble, our protein is soluble but strongly
> degraded by proteases. We tried to reduce levels of IPTG or the
> temperature of expression, nothing worked. The interesting thing is that
> in the absence of IPTG the protein looks undegraded and even lowest
> concentrations of IPTG cause its degradation without increasing the amount
> of a full length product. I do not think there is a cure for the problem
> in this E. coli strain. We use BL21(DE3) from Novagen that is supposed to
> be low in some proteases. 
> Do you know of any other strains that may be suitable for the problem?
> As our gene cloned in a variety of vectors there is no absolute necessity
> of T7 polymerase.
> 
> Simon Hoffenberg
> Baylor College of Medicine
> Houston, TX
> simonh at bcm.tmc

Hi Simon

  Try inducing for a short time at room temp (you can lower the temp just
before induction).  If that doesn't help, you can try adding PMSF to 0.5
mM during the induction.  Try a 1 hour induction without PMSF and then
another hour after adding PMSF.
  The protease missing in BL21 is the ompT protease which can be a real
beast for some proteins.  I am not aware of commercially available DE3
strains lacking other proteases but someone must have tried to make such a
strain.

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-635-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-635-68-16
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________



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