protein expression in PLysS

ChenHA hachen at bc.ic.ac.uk
Mon Mar 29 12:51:12 EST 1999


On Sun, 28 Mar 1999, Kelvin Luther wrote:

> Anant Patkar wrote:
> > 
> > We have also observed leaky expression in E. coli BL21(DE3) with
> > pET23-based
> > vector.  There was also significant plasmid stability problem.  Because
> > of the leaky expression, only 60% of the cells would have plasmid after
> > reaching stationary phase.  Has anyone seen this?
> >  
> I have seen my expression drop off  continually.  I have to keep
> reclaiming my original transformants from -70 c to get the original
> levels.  I tried the plasmid stability test in the pET manual and got no
> useful information.  I have also tried retransforming with the original
> plasmid sample, but have not been able to regain suitable expression
> that way.  

pET23 vectors do not have a lac operator site for lac repressor to
bind, therefore these vector tends to be leaky.  If you have a protein
that is toxic and has a problem with plasmid stability, it would be
advisable to use vectors that has a lac operator such as the pET21
series which make them more tightly controlled as they are doubly
repressed (doubly because the T7 polymerase is also controlled by lac 
operator).  Otherwise you should use cell that has pLysS or pLysE, but
I tend not to use them as they tend to cause more problems from my own
experience.

It is generally good practice to freshly transform your cell with
pET plasmids for every expression as they seemed not to be
always stable in frozen stock.  





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