pET15b (Novagen) IPTG induction

Ho-Leung Ng via methods%40net.bio.net (by holeung from berkeley.edu)
Fri Sep 5 13:34:51 EST 2008


Some constructs just won't express in E. coli, especially if it's
eukaryotic in origin. My first suggestion is to try the expression at
room temperature. Otherwise, change the construct, such as adding a
fusion (His, MBP, GST, etc.) tag. Another possibility is that your
protein is toxic. You can check by seeing if there is cell lysis or
poor growth.


ho

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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



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