Q. About Protein Expression !!!

Boris Steipe steipe at LMB.uni-muenchen.de
Mon Mar 17 07:48:03 EST 1997


Orhan Ertughrul wrote:
> 
> In article <5g41ra$c94 at ustsu10.ust.hk>, boella at uxmail.ust.hk (Miss ELLA
> CHAN) wrote:
> >
[...]
> >   I have been heard that we can start the induction at OD600=0.6, that
> >   is the pre-log phase of the growing bacteria or we can do it at
> >   OD600=1.5 which is the late log phase. Which condition should be the
> >   best?
> 
> I'm working with a pET 11d expression system in JM109 DE3, my protein is
> not expressed in large quantities unless I induce at an OD600 of 1-1.5.
[...]

T7 RNAP promoter based expression systems (like pET or pRSET) in our
hands definitely are different from "normal" lac or tac based promoters.
While growth and expression continues under E. coli RNAP promoters (thus
we induce around OD600 = 0.7 - mid log phase), there is no more growth
once the T7 polymerase is induced (thus we induce at OD600 above 1.0 -
late log phase), there is simply more cell mass). Once the cells have
their synthetic machinery turned off, expression will not work very
well. Thus, as a rule of thumb: earlier is better. 

Cheers,
Boris



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