problems in protein expression in E.Coli

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Mar 3 23:43:51 EST 2000


On Wed, 1 Mar 2000 22:54:41, tao jiang <tjiang at bcm.tmc.edu> wrote:

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

You don't say whether you have optimized your expression system for yield or 
for solubility of the expressed protein. Since every protein is different, you
need to optimize the system. Here are some suggestions: 

1- your gene is under control of the T7 RNA polymerase (not lac) promoter. The
IPTG is used to induce expression of the chromosomally-integrated T7 
polymerase gene. 

2- Growth of cells: grow a 3-ml overnight culture, innoc 1 mL into 50 ml, grow
to mid-exponential phase (0.3 A600), then innoculate into 1 liter broth 
already at 37 deg. In general, bacterial cultures grow more "evenly" when 
diluted no more than 1/50X each time. 

3- Optimization of yield:

   a- do several small scale cultures (50 mL in a small flask). Innoculate 
with 0.5 mL of mid-exp phase cells. Induce different flasks at A600= 0.3, 0.5,
and 0.7. Grow at 30 or 37C and do the induction at the same temperature.

   b- For each flask, take 5-mL samples just before induction, and at 1 hr, 3 
hr, 6 hr, 12 hr, and 18 hr after induction. Pellet each sample, resuspend in 
cracking buffer (SDS lysis buffer), boil, and freeze until all samples are 
collected. (You could freeze either the cell pellet or the SDS lysate, 
preferably at -70 C.) 

  c- Reheat & load a portion of each sample on an SDS gel. This tells you the 
optimum cell density for induction, and the time course of induction. 

  I anticipate that induction will be best at A600=0.4 to 0.6, and for 3 to 6 
hr. If the cells are grown close to stationary phase (I've never used T-Broth,
but maybe 2-3 A600 is max), induction will be poor, and the expressed protein 
will be more like to be proteolytically degraded. Cells in stationary phase 
typically "turn over" nucleic acids & some protein to permit de novo 
synthesis. 

4- Optimization of yield: 

  a- Using optimum conditions from above, vary the concentration of IPTG, and 
the time and temperature of induction to see what gives best percent folding. 

  b- Same 25-mL scale, but lyse cells by sonication (or whatever you do now), 
separate into supernatant & pellet, resuspend pellet in SDS buffer, and load 
both supt and pellet onto SDS gel. Comparison of lanes tells you the % of 
protein in the soluble fraction. If you know that heme is required for 
folding, make sure the precursor you add can be transported across the E. coli
membranes. 

  c- Grow 6 to 9 50-mL cultures in parallel, say at 30 or 37C, but 30 min 
before induction reduce temperature to: t1=15C, t2=20C, and maybe t3=25C, 
using 3 flasks at each temp. (Most papers that report better solubility at low
temperatures cite 15-20C.)

  d- At each temperature, try IPTG at 25, 100, and 500 micromolar IPTG (0,01 
to 1.0 mM). Follow the time course for 0 hr to 24-48 hr post-induction. The 
lower the IPTG, the longer the time. 

I've read one credible paper in which almost complete solubility of yeast 
phosphorylase b was obtained by inducton with 25 uM IPTG for ~1-2 days (forget
the temp). 

You may aleady know enough to bypass some of these steps. We don't ever do all
these (though if I were teaching a new student, I certainly would!). 

Hope this helps. 

  

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| Dr. Peter Gegenheimer       | Vox: 785-864-3939  FAX: 785-864-5321   |
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