More pET problems

[JG. Head]

Hi,

After the very helpful response to my last query (thanks!) I have decided 
to post another message and see if anybody can help me with the latest 
hitch in my pET expression.

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.

Thanks in advance,

Jared


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Jared Head     at the Department of Biochemistry, University of Bristol