I've been trying to express two proteins in the BL21(DE3) strain of E. coli.

One gene is in a pCR2.1 vector and the other in a pET-expression vector. When I induce with IPTG and run on an acrylamide gel I only get the protein encoded in the pCR2.1 construct. If I transform the BL21(DE3) cells with each vector independently (only one of the plasmids in the cells) I can get expression for either protein.

Does anyone have any Idea why I wouldn't be able to induce expression from the pET vector in the double plasmid strain but can induce expression from either vector in single plasmid strains?

  • 1
    $\begingroup$ I edited you post in a way that I hope makes things a bit clearer. ——— Some suggestions for edits to your post that may help you get a better answer would be to include answers to the following questions: 1) How are you ensuring that both plasmids remain in the co-transfected cells? 2) What are the proteins you are expressing and are they known to interact with each other in any way? 3) Would the way you prepare your protein samples for PAGE effectively solubilize proteins that were in inclusion bodies? $\endgroup$
    – tyersome
    Mar 19 '20 at 2:35
  • $\begingroup$ 1) the use of two antibiotics kanamycin and chloramphenicol, 2)the proteins being expressed are NPR1 in the pCR2.1 (protein related to systemic acquired resistance in arabidopsis) and TGA3 a transcription factor in the pET vector. The two proteins have been shown to interact in Y2H. But didn't interact in the pull down assay since the TGA3 wasn't induced. 3) I don't think it has anything to do with insoluble aggregates since in the single plasmid strain we see the TGA3 protein. Thanks @tyersome . $\endgroup$
    – jack
    Mar 19 '20 at 2:47

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