• Maybe your flask cultures are better aerated
• Your M9 doesn't contain glucose, correct? If it does then maybe the flask culture is allowed to eventually exhaust the glucose and overcome catabolite repression whereas the fed-batch culture continually receives it.
• Does your broth for the fed-batch culture include antibiotics and the inducer?

Expression using fed batch involves many factors..

1. Inoculum %: the percentage inoculum which is giving better expression in flask might not give in fed batch fermenter..(what is the volume of the fermenter.?)

2. whether you are looking for extracellular secretion or intracellular. If extracellular then u might have to consider the stability of ur expressed protein in fedbacth as factors like extra cellular proteases could play some role.

3. Checking the expression in initial inoculum..? I generally used to do this, The culture inoculum which goes into the fermenter must be checked with PAGE or assay based if the expression of ur desired protein is present and then give the inoculum. this will allow you to eliminate false positive (contamination) in inoculum.

4. M9 salts is a minimal medium, to my knowledge if its a heterlogous expression using e coli, using minimal medium will not give u much yields as the rate at which  total cell mass growing in fermenter will, be very low. A rich medium like LB will allow more cell density and should give better expression.

5. Flask vs fermenter. What works in flask (yield of protein) need not necessarily work in fermenter and vice versa.. I did experience same yield of protein both in flask work and fermenter using Bacillus expression system.

thanks Goutham,

the protein is asparaginase and intracellular and we get good expressions in erlene-meyer flask cultures both on M9 and LB. I'm also sure about the absence of contamination.

• Maybe your flask cultures are better aerated
• Your M9 doesn't contain glucose, correct? If it does then maybe the flask culture is allowed to eventually exhaust the glucose and overcome catabolite repression whereas the fed-batch culture continually receives it.
• Does your broth for the fed-batch culture include antibiotics and the inducer?

If you've considered everything about your expression system and it all makes sense and you're confident no mistakes were made and should function as expected then all I can say is that increasing cell density can have inhibitory properties on protein expression. I've seen this in my own work.

Is the pET system repressed by presence of glucose?

Thanks everybody, the problem was from the plasmid stability.