I have been using the Duet system from Novagen (or whatever it is called
these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of
my proteins did not work in either vector. Either, one protein expressed
or the other. I played around with the promotors (they are both T7) by
changing one to the tac promotor. This increased the expression of this
gene but shut off expression of the other. The only way I could get my
proteins to co-express was to use pGEX vector with one protein, and
pRSFDuet with the other protein (leaving the second MCS empty). There is a
paper which sums up co-expression in E coli.
http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html
There is really absolutely no problem co-expressing two proteins both from
near-identical pET vectors as long as the two plasmids carry difference
selection marker. We've used pET24 in combination with pET28 or pET31 on
several complexes and it always works as long as you keep both antibiotics
around.
- Dima
