Hi!
I suspect that a protein expressed in E. coli undergoes some posttranslational modification due to the cleavage of some protease. Therefore, I have thought of studying its expression during bacterial growth by adding some broad-spectrum protease inhibitor. My question is if, by adding a general inhibitor of those used to add cell lysate to avoid protein degradation during the purification process, it will penetrate inside the bacterium if I add it to the culture medium.
What other alternative could I use?
Thanks in advance
Your strategy probably wouldn't work as protease inhibitors are also quickly inactivated by water. If you want to slow down protease activity in cell cultures, grow your recombinant cells suspension shaking cold at 16 Celsius. Protein folding, cell division, proteolytic activity and all cell processes are slowed down at colder temperatures. You can grow your cells even colder. It just takes longer.
There are also E.coli expression strains that lack certain proteases.
https://www.neb.com/faqs/0001/01/01/what-are-the-strain-properties-c2566#:~:text=Protease%20Deficient%20(%5Blon%5D%20ompT,surface%20of%20wild%20type%20E.
These enzymes cannot be inhibited and their action can be slowed down by lowering the temperature
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