problems in protein expression in E.Coli

Carmelo carmelo.diprimo at bordeaux.inserm.fr
Thu Mar 2 03:33:12 EST 2000


In article <38BD9FB0.4B7260B4 at bcm.tmc.edu>, tao jiang <tjiang at bcm.tmc.edu>
wrote:

> Hi, I am new here. I hope I can get some help from you.
> I am doing some work on inducible Nitric oxide synthase (iNOS). I have
> the BL21(DE3) strain transformed with plasmid containing cDNA for iNOS,
> which is under control of lac promotor. I am trying to purify the inos
> from this BL21(DE3). But I always got fluctuating iNOS activity ( I mean
> sometimes high and sometimes low, and so does the  inos protein verified
> by western) in bacterial lysate. Since the starting point is not stable,
> I can't get good result after several steps of purification. I wonder ,
> for recombinant protein expression in E.Coli, is there any special tips
> in bacteria incubation? I do like this: inoculate a
> -70 centigrade bacteria stock to a 1 liter terrific broth, 37C 250rpm
> overnight until the OD600 to 1.0, cool it down to 25C, add IPTG to 1mM,
> and also some heme precursor for inos folding, then avoid light shake at
> 150rpm room temperature for another 24 or 48h.
> Does anybody has comment about these procedures?
> And for bacteria protein extraction, did anybody use Pierce
> product:B-per or B-per II, do you have any suggestion on that?




Hello,

I am not familiar with NOS but years ago I used to purify p450cam from
cloned E. coli. I had problems with the expression of the protein too at
the begining.
One thing really improved the expression. Each time I decided to start an
expression I used freshly transformed bacteria and not the stock from
-70°C. In my case this was the major key to really improve the expression
of the protein. It's a little bit more time consuming but once you have
your plasmid from the miniprep and your competents which are stable about
1 month that is not such a big deal.

The other thing I used to do is instead of directly inoculate my 6 L erlen
(2.5 L of LB), first I inoculated 50 mL with the freshly transformed
bacteria and then after 2 or 3 hours (in logarithm phase of the growth)
the flask with 1 mL of the 50 mL and then one night at 37°C. Shaking at
150 rpm sounds OK to me.
Why after adding IPTG room temperature and not back 37°C? Anything to do
with IPTG? I'm not sure heme precursors are necessary, I never add that
for p450cam and it worked just fine. I have no idea about NOS of course!

One other to keep in mind, it seems that hemes proteins do not like oxygen
at least when expressed in E coli. Each time I tried to run the
fermentation in big fermentors with strong oxygenation, I had great
amounts of bacteria but almost nothing on protein expression. Actually I
did not spend time to understand that because the expression was just fine
in erlens.

Hope this helps

Carmelo

ps : I would not add IPTG at the end of the growing phase (I am not sure
that after one night the bacteria are growing a lot more) but rather
during the logarithm phase. It makes really more sense to me




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