E. coli proteolysis

shi tao tshi at ucla.edu
Sat May 17 00:31:52 EST 1997


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 




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