fresh transformations for protein expression??

Flip Hoedemaeker fhoedem at oci.utoronto.ca
Tue Jan 9 16:51:31 EST 1996


davesmith at bioch.tamu.edu wrote:

> Torsten,
> We routinely use BL21 (DE3) harboring various constructs, all of which
> are pET11a derivatives of one sort or another.  All of our expressions
> with this system to date have been membrane proteins (lysis proteins
> from bacteriophage).  We have seen the effect you mentioned
> consistently since first using the system.  We rationalize this effect
> to 1) the toxicity or nature of the protein being expressed and 2) the
> dual layer of transcriptional control (repression at the promoter for
> T7 RNAP and at the hybrid promoter on the vector), although much
> tighter than standard cloning and expression vectors, IS LEAKY.  In
> other words, in our experience, E. coli has difficulty with membrane
> protein overproduction, and the pET system is never OFF.  Thus, any
> colonies on a plate are almost guaranteed to have a low background
> level of plasmid-encoded protein.
[..]
> Is anyone aware of other problems which we haven't considered?
> Does anyone have any suggestions on how to alleviate the need to use
> fresh transformants for every experiment?  I'd sure like life to be
> easier!
> 
> Dave Smith

If you think leaky expression is the problem in your case, you should
try the pLysS variant of BL21(DE3). The small amount of T7 lysozyme
produced inhibits T7 RNA polymerase, decreasing leaky expression. If
this doesn't work, try pLysE, which makes more lysozyme. Alternatively,
try other (DE3) hosts, some people claim they can better control
expression in e.g. HMS 714 (DE3)

Hope this helps, Flip



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