fresh transformations for protein expression??

Kaj Stenberg kstenber at cc.Helsinki.FI
Mon Jan 29 05:28:56 EST 1996


David Goodin (dbg at scripps.edu) wrote:
> Yes, I have also seen exactly this behavior for years in a
> t7 expresion system for a nontoxic protein expressed at high
> levels. You can even take an old culture and restreak to 
> reselect resistant clones and it still wont express. However,
> I can take that culture, isolate the plasmid, transform,
> and expression is back. We see significant plasmid loss in
> cultures even with heavy and continual amp selection, but
> this may be something else due to the restreak phenom.
> I don't know what it is... Only idea is that once cells
> begin to express they grow slowly, but produce enough
> lactamase to quickly degrade the amp in the media, then
> quickly growing untransformed cells take over. Anyone have
> a better undertanding?

I used an amp-resistant pET3d-derived plasmid. In my case I have a very
long induction time (16-20 hours, believe it or not!), and I have
assumed that any amount of amp added will be degraded practically
immediately. I Inserted a kanamycin-gene into the amp, and the yield
rose dramatically. However, I too seem to simply lose expression upon
storage (still kan-resistant!), and I have with great interest followed
the discussions here. But does anybody have a citable reference? The
earlier mentioned Bio/technology reference (vol 13 1995) didn't seem to
deal with exactly that aspect.

Hope someone can help me,


_____________________________________________________________________________

Kaj Stenberg                    tel: +46-8-7287673
MBB/structural group            fax: +46-8-327626
Karolinska institutet           e-mail: Kaj at xray.bmc.uu.se
17177 Stockholm                         Kstenber at plootu.helsinki.fi
Sweden                          URL: http://gamma.mbb.ki.se/~kaj/

______________________________________________________________________________





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