More pET problems

JG. Head bijgh at
Tue Aug 8 08:36:17 EST 1995


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 Head     at the Department of Biochemistry, University of Bristol

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