E.coli and toxic proteins

Dr. Duncan Clark duncan at genesys.demon.co.uk
Wed Jul 3 04:04:43 EST 1996


In article <genecutl-0207961429490001 at kosh-161b-2.berkeley.edu>, gc
<genecutl at mendel.berkeley.edu> writes
>I am failing at my attempts to express a recombinant protein 
>in E.coli.  The vector has a T7 promoter as well as a lac operator
>and repressor, and I have tried using BL21 and BL21/pLysS for the
>expression.  The problem is that the cells won't grow once the plasmid
>is in them.  They will grow on a plate, actually, but once I put them
>in liquid culture they don't grow at all.  I have tried growing a lawn
>of cells on a plate, then scraping that off and resuspending them in
>LB, then inducing, but I got no protein production.
>Does anyone know any tricks to get a toxic protein expressed in E.coli?


Your problem is that you haven't managed to completely repress the
expression ie there is sufficient T7 RNA pol being expressed before
induction which leads to expression of your protein which is very
toxic/lethal. An alternative may be to grow your clone in liquid in say
BL21 (not DE3) and infect it with CE6 which is a lambda clone containing
the T7 RNA pol. That way you should at least get a reasonable amount of
cells.If possible I would also try and use a plasmid which is kan not
Amp resistant. With your toxic gene product at least with Kan you stand
a good chance of retaining your plasmid as the culture grows unlike Amp.

Alternatively you need to find a better plasmid, in terms of more
repression, for your clone. There are a number which have one or more
lac operator sites between the t7 promoter and the rbs. This may or may
not help. 

It's a difficult problem.

Best of luck.

Duncan 
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My mind's made up. Don't confuse me with the facts!
Duncan Clark
GeneSys Ltd.




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