pET15b (Novagen) IPTG induction

I am having trouble with inducing expression of a protein that I have
cloned into pET15b (Novagen). I have tried induction in BL21 E. coli
with 1.0 mM IPTG in LB/100 ug/ml Ampicillin, at an OD(600 nm) of
0.5-0.6, at 37 C for up to 8 hrs and then overnight. Harvested cells
are lysed by 30 min. incubation with 1 mg/ml lysozyme, followed by
sonication (6x 10s for small scale 4 ml lysates, prepared from up to
100 ml cultures). But so far, my IPTG-induced lysates on SDS-PAGE look
just the same as a non-induced control; I see no IPTG-dependent band at
52 kDa where I want to see one. I have re-sequenced my plasmid after
transformation into BL21, and the sequence is correct. Am I missing
something somewhere?

Any enlightenment would be greatly appreciated

Paul Phelan
Tufts University
Department of Biochemistry
Boston

In article <[Only registered users see links. ].net> , "Paul J. Phelan" <[Only registered users see links. ]> wrote:

If the above is really correct, you won't see induction. BL21 cells
don't have T7 DNA polymerase required to read off T7 promoter
found in pET plasmids. You need to use BL21(DE3) strain that
has replication-deficient T7 phage in its chromosome. Or any other
(DE3) strain.

If you did use DE3, there are several other options like protein
found in inclusion bodies (give milky appearance after cell
lysis) or protein being cytotoxic and cells expressing it being
selected against during overnight culture due to the slightly leaky
promoter. In which case the overnight must be done in rich defined
medium containing glucose (MDGA, for example; part of the
autoinduction protocol).

DK

plates

On Sep 4, 8:25*pm, "Paul J. Phelan" <[Only registered users see links. ]> wrote:

Hi Paul,

You want to use BL21(DE3) cells and not just BL21. The former cells
contain a ë prophage carrying the T7 RNA polymerase gene and lacIq.
When you add IPTG to the culture, it binds to the lac repressor coded
by the lacIq, allowing expression of the T7 RNA ploymerase which in
turn binds to the T7 promoter on the pET plasmid and turns on genes
downstream of the promoter.

Good Luck!