Problems with expression in E.coli

Paul Klosen klosen at neurochem.u-strasbg.fr
Thu Mar 22 08:13:01 EST 2001


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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