We have also had success expressing two proteins separately and then mixing the soluble fractions of the lysates in the presence of a complex-initiating ligand, where one protein has a His-tag (preferably the protein that expresses to a lower extent) and the other no affinity tag followed by a Nickel affinity column to capture the complex (unpublished).
Laurie Betts UNC Chapel Hill On Mon, Jul 16, 2012 at 11:55 PM, Biswajit Pal <p...@ccmb.res.in> wrote: > 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" <klenc...@facstaff.wisc.edu> > To: CCP4BB@JISCMAIL.AC.UK > 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 >
