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pET15b (Novagen) IPTG induction
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Paul J. Phelan
Sep 4, 2008, 11:25:47 PM9/4/08
to met...@magpie.bio.indiana.edu
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?
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
Ho-Leung Ng
Sep 5, 2008, 2:34:51 PM9/5/08
to Paul....@tufts.edu, met...@magpie.bio.indiana.edu
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.
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
-----------------------------------------------------------------------------------------------
E. Lis
Sep 8, 2008, 7:00:24 AM9/8/08
to met...@magpie.bio.indiana.edu
It is possible that your protein is present in inclusion bodies, and
lysozyme and sonication will not disrupt those. Have you tried lysis by
8M urea?
You could also try running entire bacterial cells suspended in SDS-PAGE
sample buffer (as 10 min in sample buffer at 100^C should do the trick).
lysozyme and sonication will not disrupt those. Have you tried lysis by
8M urea?
You could also try running entire bacterial cells suspended in SDS-PAGE
sample buffer (as 10 min in sample buffer at 100^C should do the trick).
Message has been deleted
vinod...@gmail.com
Sep 17, 2008, 4:55:35 PM9/17/08
to
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!
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