Problematic Basal Expression Levels

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wzhang
wzhang's picture
Problematic Basal Expression Levels

I am currently trying to purify a GST-fusion protein from E. Coli. The first small-scale trial after transformation of the E. Coli yielded stunning, nearly ideal results. The induced samples yielded generous portions of protein, while the uninduced samples produced very little.

I am incubating the E. Coli to an optical density of .6-1.5 before induction. Then, IPTG is added to a final concentration of .05 mM. It is incubated at 37 degrees C for three hours, with vigorous shaking (200 rpm).

For the second trial, I tried to extend the first trial's success to a large-scale purification. This failed. There was very little noticeable difference between the induced and uninduced cultures. I guessed that it may have been a problem with the basal expression level, so I tried two more large-scale experiments using 2xYT medium containing 1% and 2% glucose. Nada.

Then I wondered if it was a problem with the cells, that perhaps only the first-generation newly transformed E. Coli were able to effectively express the protein. I transformed a new batch of cells and tried again. Once again, very little difference between the basal and the induced expression levels.

I am going to try going back to growth medium without glucose and see if the glucose could have inhibited protein expression in any way. I also have hunches that the protein may be toxic to E. Coli, limiting the amount that can be produced, or that the protein may be being degraded in the large-scale trials. However, I have no feasible explanation for why this did not occur in the first small scale trial. I'm quite stumped. Any insights, comments, or suggestions would be greatly appreciated.

R Bishop
R Bishop's picture
wzhang,

wzhang,

Wow that is too bad. In this situation, you must have done something different in the small scale experiment. Have you changed any key reagents? Bought new supplies? There must be one thing that changed in the scale up. Typically I always start by analyzing the big three. Time, temprature, and concentration. Unfortunately in your situation, you got 2 things to deal with the bacteria and the IPTG. Were your cultures made fresh and split at the right times? I had a similar experience, when I got lazy and tried to scale up with bacteria that were in lag growth phase.

Keep us posted with your results and WRITE down everything you do.

RB

kriodos
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are you sure that in the

are you sure that in the first time you get the real protein? did you check the protein obtained is not a contamination of one of your labmates? After the second large scale transformation and indcution whereas you don't get anithing, Di you check that the conmstruction have the rigth secuence? My advice.
a)Check construction
b)Transform Bl21 cells
c)"wash" the petri plates with the bl21 transformed cells and use as inoculo of the lage scale purification. At least 1 plaque (50-200 colonies) per half liter of culture plus the antibiotic.
d) induce with 1mM IPTG
If this dosn't work you are in a throuble
good luck

Add colour 2 ur life
Add colour 2 ur life's picture
hi

hi

Yes,I agree with Bishop. Temparature,Inducer,time and media are the key elements for bacterial expression.

increase the IPTG concentration to 0.5mM instead 0.05.
After induction reduce temparature to 25-30Degree and keep it for overnight.

While doing your analysis make sure that you are keeping od normalization/pellet:lysis buffer ratio same with and without induced sample.

better to normalize the od to 10 both and analyze it may tell exact expression level.

All the best.