Induction problems with pETsystem - (Jun/06/2001 )

I'm using pET expression system to overexpress a flavoprotein. After 3h of induction with IPTG at 37ºC I have an extremly high level of flavoprotein production without IPTG compared with the basal expression of Escherichia coli. If someone has any idea about what may be happening or has had the same problem, please get in touch. Thank you very much.Manuela Broco

I've had the same problem before and was equally frusturated. I found out, that somehow the E.Coli strain that I was using was contaminated by another strain, and I was expressing a completely different protein, which could not be induced by IPTG.

This may not be your case, I would repeat my transformation and make absolutely sure that you are using BL21 competent cells...is it possible you transformed with non-protein expression cell line? You would still see protein expression, but not massive induction.

Also, I would induce longer. I've induced bacteria for 9 hours before, to get absolute HIGH levels of protein. I would run a series of tests, where you induce your bacteria...remove 0.5 ml of bacteria soup every hour for at least 9 hours and run it on an acrylamide gel. (Boil it for 45 sec.) Compare expresssion from 1 hour to 9 hours against non-induced bacteria.

I've had the same problem before and was equally frusturated. I found out, that somehow the E.Coli strain that I was using was contaminated by another strain, and I was expressing a completely different protein, which could not be induced by IPTG.

This may not be your case, I would repeat my transformation and make absolutely sure that you are using BL21 competent cells...is it possible you transformed with non-protein expression cell line? You would still see protein expression, but not massive induction.

Also, I would induce longer. I've induced bacteria for 9 hours before, to get absolute HIGH levels of protein. I would run a series of tests, where you induce your bacteria...remove 0.5 ml of bacteria soup every hour for at least 9 hours and run it on an acrylamide gel. (Boil it for 45 sec.) Compare expresssion from 1 hour to 9 hours against non-induced bacteria.