More pET problems

Ted M. tedm at darkwing.uoregon.edu
Fri Aug 11 23:28:11 EST 1995


In article <DCzv4I.Ds1 at uns.bris.ac.uk>, bijgh at zeus.bris.ac.uk (JG. Head) wrote:

> 
> I have my 200aa insert safely ligated into pET 15b, and while I have been 
> getting expression of my peptide in BL21(DE3)pLysS I was having problems 
> with chloroamphenicol acetyl transferase (expressed by the 
> pLysS plasmid) co-purification so I've put my pET into BL21(DE3) instead. 
> 
> I have two problems:-
> 
> 1)  The expression of my protein in BL21(DE3) seems to be "negatively" 
> inducible.  I get constitutive expression of the protein, until I add 
> IPTG and then expression stops.  It's not a big problem, but it is puzzling.
> 
> 2)  More importantly I only get expression of my protein when I grow up a 
> small amount.  I've just grown up 5mls and 500mls of my cells identical 
> in every way apart from volume, and the small sample has over-expressed 
> my peptide, while in the large sample there is nothing.
> 
> Has anybody any ideas what's going on?  Any suggestions would be very 
> gratefully received.

> Jared

It seems as if you have a toxic gene product, Jared. The pLysS (and pLysE)
were designed to down regulate the T7 RNA pol which leaks from the DE3 
gene. Thus your expression was more controlled with the pLysS than with
the plain DE3. Typically the growth stops when a toxic gene product is
turned on, this may appear like expression stops. If constitutive (low
level) expression won't cut it you may have to change around your system.
The problem 2 is probably because your plasmid is gone with the large
scale cultures; remember all the selective pressure (Amp) is gone within
about 1-2hrs, you could try replica plating to see if this is the case.
Refer to the original pET paper of Studier (Methods of Enzymology) to see
what to do with very toxic gene products. I would attempt to transform
your plasmid into plain BL21, no DE3 and then follow a conservative seed
train to get higher cell densities and culture volumes, then infect with
the phage which supplies the T7 RNA Pol. I belive the phage is
commercially available. This should give your gene product a fighting
chance. If still troubled a pulse chase exp. may let you see whats
happening. Good Luck.

regards,

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at Darkwing.uoregon.edu

PS will you be Head of the dept. soon?...I couldn't resist...sorry



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