Problems with expression in E.coli

Paul Klosen klosen at
Fri Mar 23 13:16:01 EST 2001

Sergio wrote:
> I had a couple of clones on a HisTag vector which expressed poorly. After
> several months of working with them and sending them from lab to lab, they
> didn't express at all (only the original clones worked). I switched to MBP
> fusion system and expression (and specially solubility) are much better, and
> purification are simpler and needs less whasing steps (and no imidazol to worry
> about). There is an obvious disadvantage: the size of the quimeric protein, but
> depending on what you want to do with your sample it could be irrelevant.

The proteins I am trying to express wil definitely not be soluble in
bacteria. They will most probably end up in inclusion bodies and will
have to be purified under denaturing conditions (8-9 M Urea). To my
knowledge, only the HIS-tag works under these conditions. I performed
this type of expression/purification scheme already with intermediate
filaments and I worked fine (actually I often use some of these
constructs as expression controls because they work VERY nice).

Furthermore, I want to denature my protein and thus I don't care if my
protein is not correctly folded. All current attempts to produce
antibodies against this protein in its native state or against peptides
have failed.

Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite F-67000 Strasbourg,
Tel.  Fax.  klosen at

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