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.