More pET problems
bijgh at zeus.bris.ac.uk
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
Thanks in advance,
Jared Head at the Department of Biochemistry, University of Bristol
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