E. coli proteolysis

[plxsrt at pln1.nott.ac.uk]

tshi at ucla.edu (shi tao) wrote:

>I SAW THIS IN ANOTHER NEWSGROUP AND WE HAVE THE SAME PROBLEM.  DO YOU GUYS HAVE
>ANY GOOD SUGGESTION!  THANKS A LOT!

>Hi, we got similar problem too.  

>The protein we've been trying to express are the Dorsal protein and Groucho 
>protein, two transcriptional factors from fruit fly.  Even with E. coli strain BL 21 we still got a lot degradation products.  But we did get pretty 
>good purification for one of Groucho's deletion form.  Which GST fusion system are you using now?  Is it from Pharmacia?  
>We never tried to put protease inhibitors directly into the culture.  I wonder if it can work, but we did add them during the purification process.
>Have you consulted the company?

>--Tao Shi

>In article <mbzrl-150597154222 at pmbfjd0.nottingham.ac.uk> 
>mbzrl at mbn1.biochem.nottingham.ac.uk (Rob) writes:>From: 
>mbzrl at mbn1.biochem.nottingham.ac.uk (Rob)>Subject: E. coli proteolysis
>>Date: 15 May 1997 14:50:07 GMT

>>Can anyone help please..

>>We've been trying to express a pro-hormone as a GST fusion in E. coli
>>(DH5-alpha) but seem to be having problems with proteolysis - we think this
>>may be occuring at double basic sites but are not completely sure.

>>Has anyone 
>>a) had similar problems
>>b) been able to solve the problem by using e.g. JM105s or BL21s
>>c) tried inhibiting endogenous coli proteases (i.e. whilst they are
>>growing) by adding protease inhibitors to the broth (will they get into the
>>cells ?)

>>Thanks in advance

>>Rob Layfield 

I have seen similar problems but fortunately in the BL21 (DE3 plyS)
background the protein was stable.  You don't say what induction
conditions you have used- have you tried altering factors such as
induction temp, induction time and induction method?  

Simon T