Problems with expression in E.coli
sergioal at bbm1.ucm.es
Fri Mar 23 05:21:58 EST 2001
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.
Paul Klosen wrote:
> 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 Klosen, PhD
> CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
> Universite Louis Pasteur 12, rue de l'Universite F-67000 Strasbourg,
> Tel. 03.88.35.85.04 Fax. 03.88.24.04.61 klosen at neurochem.u-strasbg.fr
More information about the Methods