What bacteriocin are you expressing? Do you know if it kills E. coli or not? Because if it is lethal to E. coli then you are never going to recover the plasmid unless you also provide the "antitoxin" protein to let the cells survive.

If you want to use pET19 then you will need to use the NcoI site for the upstream cloning site if you don't wish to have the His tag and upstream material in your protein.

Also many bacteriocins need to be secreted and some also need accessory proteins in order to be activated, but not all. Do you know whether your protein requires anything like that?

Michael J. Benedik Thank You for answer, it is a leaderless peptide that is only active against Gram + bacteria, although I don't know if it is toxic inside the cell once expressed. I need it for structural research afterwards, the active peptide is desirable, but not needed at the moment.

I presume you know that many peptide bacteriocins are highly modified so for structural studies this might be important.

Were you able to get the correct clone in pQE or pMAL? If you had the right clone but are not seeing protein then the issue might be something different. I don't know that moving to pET will be a panacea for you. It could well be that as a short and possibly unstructured peptide in E. coli it is rapidly degraded.

Michael J. Benedik Yes, ive got in both pQE and pMAL, but out of many expression attempt only 2 times were success. I am aware that degradation is possible, but i just want to try in one more E. coli expression system if I want to assume that it is "impossible" to express in E. coli. I chose pET because there is a paper where it was described expression of a homologous protein.

As for modifications, the only modification is the N formylmethionine, as peptide is sequenced. Besides, I also have chemically synthesized protein, but without N formylmethionine, that shows ~2x lower activity than wildtype.

Did you notice any toxicity when you had clones in pQE or pMAL, such as slowing down of growth after adding inducer?

In addition to trying to create your pET clone, you might want to retransform your DNA from the pQE or pMAL clones into fresh cells and maintain them on glucose supplemented plates to reduce expression. Then screen them again and notice if there is some toxicity. It could be that the clones are somewhat toxic so you are constantly getting mutants that no longer express. Therefore it might be worth rescreening from your original plasmid stock.