I am having trouble with expression of 6 kDa long bacteriocin in pQE and pMAL systems. So i wanted to try the cell-free expression and i want to hear experiences with cell-free expression systems and other strategies for overcoming the no expression issue. I've tried different cells, different plasmids, different expression environment (inducer, temperature, time). I've got it once in both systems and then nothing, i can't replicate it...
Thank you!
Expressing a bacteriocin could be very problematic if it is lethal to the host cell you are expressing it in. So it could be that you have just selected for mutants that are inactive or fail to express, since there is a very strong selection for those if the protein is toxic.
Do you know the molecular target of the bacteriocin?
Have you tried co-expressing with its antitoxin to see if that helps?
Have you sequenced the plasmid from a non-expressing strain?
Michael J. Benedik Thank You for answer.
I don't know the target, i just know that it is active only against Gram-positive bacteria. I haven't tried co-expressing with it's antitoxin
Yes, i sequenced the expression cassette and everything was correct. Is it possible to get inactivated while it grows in culture?
It is very possible that it picks up a mutation when you grow the culture. When I asked if you had sequenced it, I was referring to sequencing the plasmid from a culture that is not expressing it to see if there are mutations that arise.
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