Hi

I recently tried the same protocol and it worked quite fine for me. Did you test "usual" culture in M9 in parallel on the same system ? It would be nice to get some more details on what you have done/tested before. Does the problem come from the switch of a standard M9 culture to the high density approach ? Otherwise, it could be that your protein cannot be expressed in minimal medium at all.

I usually do the following (standard M9 approach):

Morning: incubate 1 colony in 5 or 10 ml LB, grow at 37°C

Evening: spin down cells and transfer them to 100 ml M9 medium (I usually add minerals (Zn, Mn, Fe) and vitamins according to Jansson, JBiomolNMR 1996), 2-4 g of glucose/L, incubate ON at 37°C.

Morning 2: measure OD (should be 1.2-2), spin down cells and transfer to 1L of fresh M9 medium. Grow to a density of OD=0.8 at 37°C and induce (you may also test induction at 20°C ON)

Beate

Hello,

Another idea is change the temperature to 30ºC after induction.

Try looking to a step-by-step description of this method.

Article A Novel Bacterial Expression Method with Optimized Parameter...