Hi, I guess someone else has already had this kind of trouble before.

I was expressing some proteins in pGEX6p1 and just one protein "X" (smaller and larger fragments) have stopped to work. All other proteins are still expressing fine, but these proteins have completelly stopped to express (smaller fragment) and reduced a lot (larger fragment).

So I concluded that something wird happened here, but I could not find it so far. I tried different concentrations of IpTG, diffrent OD, medias, flasks,... and I also have transformed them again into 2 different strains using the plasmids that I had.

The sequence is fine and they were expressing a lot before, althogh the larger frag was expressing twice much as the smaller frag.

Has anyone a sugestion to cross this trouble? I am now going to clone in a different pgex vector (pgex 2TK).

in your case lower induction temp will help.

and sometimes over-induction (I mean induction time) will do reverse, so try shorten you induction time too