pET15b (Novagen) IPTG induction

[vinodasundi from gmail.com]

On Sep 4, 8:25 pm, "Paul J. Phelan" <Paul.Phe... from tufts.edu> wrote:
> 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

Hi Paul,

You want to use BL21(DE3) cells and not just BL21. The former cells
contain a λ 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!