Problems with expression in E.coli

Marcin mnwoznia at med.uni-tuebingen.de
Thu Mar 22 08:40:11 EST 2001


hi, 

i also had this problem - GST-construct was fine and in frame, but no
protein at all: neither GST nor traces of degradation also during the
preparation, but everything works fine if i express only GST in empty
vector;
i have no idea why it's like this - i just made new shorter construct and
everything was OK,

i also had other problems with GST-fusions: the protein was really nice
expressed, not degraded, but i couldn't get anything after elution with
gluthation in concentration 5-50mM - only 0.1 SDS helped, but after
dialysis proteins precypitated from the solution,


Marcin

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On Thu, 22 Mar 2001, Paul Klosen wrote:

> Hi,
> 
> I am trying to express a protein in E.coli as a HIS-tagged fusion
> protein to produce an immunogen. I have done is already several times
> but this time I have run into a major problem .... no expression at all
> !!
> 
> I cloned my protein of interest into pET16 and pET28 using the NdeI site
> and the ATG of my protein. Sequence (and experience with other
> construcs)tells the cloning should be perfectly in phase. Also, all my
> expression controls work fine.
> 
> I transformed the constructs into BL21(DE3)-RP and BLR(DE3). They grow
> fine, have the plasmid but upon addition of IPTG .... nothing, nada ...
> Growth rate slows down a little but that's about all. I checked for the
> presence of the plasmid both before and at the end of the induction.
> 
> Has anyone run into similar problems ??
> Or has anyone an explanantion for this ??
> 
> Thanx at lot in advance
> 
> Paul
> -- 
> Paul Klosen, PhD
> CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
> Universite Louis Pasteur  12, rue de l'Universite F-67000 Strasbourg,
> FRANCE
> Tel. 03.88.35.85.04  Fax. 03.88.24.04.61  klosen at neurochem.u-strasbg.fr
> 
> 

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