Fusion protein expression in E.coli. C.Baker

Anonymous rkporter at mail.tcd.ie
Mon Aug 14 05:24:36 EST 1995


I'm attempting to produce a eukaryotic 70kDa membrane protein as a GST
or Hexahis fusion in E.coli under the tac promotor. Induction even by 
low concentration of IPTG (0.25-0.4mM) proves lethal and on 
purification by gluthatione affinity chromatography I get a range of 
sizes from 80-26kDa which collapse into the 26kDa GST protein on 
thrombin treatment. Purification is carried out in the presence of 
Boehringer Mannheim protease inhibitor cocktail with PMSF supplemented.
Could this be a problem with internal proteolysis? or perhaps 
transcription or translation?
I've tried a few strains but am now using BL21 DE3 pLys S primarily 
because its a protease mutant but I'm a bit worried because the 
plasmid does not employ the T7system.
The Ni-affinity product under denaturing conditions (8M urea) yielded 
an approx. 30kDa band. Would it be wise to incorporate a detergent 
during this procedure?
Westerns of whole E.coli with an anti-peptide antibody do not yield 
any significant bands in either case.
I would be most grateful for any advice via e-mail to 
crbaker at mail.tcd.ie
Also is there a general method to determine if inclusion bodies are 
formed and if so to isolate them?
Thanks,
C.



More information about the Methods mailing list