I fully agree with Dima. We are able to co-express and purify two interacting partners using pET28 and pET21 in E. coli. Some related references are :
J. Mol. Biol. (2011) 405,49–64 J. Biol. Chem. (2006) 281, 26491–26500 J Struct Biol. (2011) 175(2):159-70 Biswajit ----- Original Message ----- From: "Dima Klenchin" <[email protected]> To: [email protected] Sent: Tuesday, July 17, 2012 5:16:34 AM Subject: Re: [ccp4bb] co-express two proteins in E.coli >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
