Overexpression of a protien (fwd)

[Zophonias O. Jonsson]

In article <tomasd-2710951215230001 at fp5-9n.domain>,
tomasd at bdg10.niddk.nih.gov (Tomas Drgon) wrote:

> In article
> <Pine.SOL.3.91.951025101344.12308B-100000 at welchlink.welch.jhu.edu>,
> tlu at WELCHLINK.WELCH.JHU.EDU (Natalie Lu) wrote:
> 
> 
> > I have problems to overexpress my protein. 
> > The DNA sequence of the cloned plasmid is 
> > perfect. The insert was cloned into the NdeI site of the vector. Then,the 
> > the plasmid was tramsformed into BL21(DE3) cells. The clone was picked to 
> > test the overexpression of the protein. I didnot see any overexpressed 
> > protien at all.

As has been pointed out there are various reasons for T7 constructs not
expressing the wanted protein properly.  One of the more common reasons i
toxicity of the protein.  Changing the vector and using fusions are all
valid Ideas but for starters it may be easier to try a strain that allows
less "leakage-expression" than BL21(DE3).  Try BL21(DE3)pLysS or pLysE if
you can get your hands on them.

Good luck

Zophonias

_____________________________________________________________________
Zophonias O. Jonsson
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-75
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich
Switzerland
_____________________________________________________________________