Problems with expression in E.coli

[Paul Klosen]

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
-- 
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