By not expressing do you mean "not an obvious band on a Coomassie stained gel" or "No band on a Western blot"? Just asking because some proteins express poorly in spite of the RNA being highly over expressed and this can result in the band being faint and difficult to distinguish over random E. coli proteins on a gel. A Western blot (especially with an HRP conjugated secondary antibody) on both soluble and insoluble fractions is much more sensitive. You should be able to get good monoclonal antibodies against either the 6X-His or the S-Tag (depending on where you cloned your gene) if you haven't already.

Have you tried changing the temperature and time?

Alexandra Johnson I mean, not an obvious band on a coomassie stained gel. I haven't done a western blot for the same. I will check it by a western blot.

Pardhipa Nandigam I have tried inducing the protein at 37deg for 3h also. How do we decide for the particular range of temperatures to be used for checking the induction? I have used these two temperature with respect to the already available data.

What is your OD at the time of induction? Maybe increase the induction time for 37 degrees and check.

I'd start troubleshooting from the beginning--what's the origin of the proteins, why are you co-expressing, have you tried expressing one at a time, has anyone ever expressed those proteins in a bacterial system...

And then, was that gel of purified protein, lysate, or induced cells (so technically also lysate, but prepped via hot lysis)? If your recombinant protein runs similarly to host proteins, it might be not clearly visible in a lysate gel. WB is definitely better as mentioned above, but also not bullet-proof.

Pardhipa Nandigam I have done the induction between 0.5 OD/ml-0.6OD/ml.

Katarzyna Radziwon The proteins are of fungal origin and I wanted to do an invitro interaction studies of these that's why Iam doing the coexpression experiment. I have same genes cloned to a different vector as a single insert and those are expressing very well in bacterial system. However, the genes cloned into the duet vector are not expressing.

I have run uninduced cells, induced cells, sonicated supernatant, sonicated pellet on SDS-PAGE gel for the comparison. I also did a pull down against the tag however elution did not have the desired proteins. This is how I concluded that the proteins are not expressing.

Any chance they are interacting in a way that masks the tags? Do you have structures of those proteins? Did you do alpha fold of the complex?

Katarzyna Radziwon there is no crystal structure details available for these proteins. I didn't try the alpha fold structure. I will give it a try. Do you think by any chance these proteins are getting degraded in the bacterial cell after expression due to which it is not detectable?

If they express just fine in single constructs, the degradation wouldn't be my first guess. If possible, share a gel from expression of single constructs and from expression of duet, it's always easier to form hypothesis when you actually take a look at the data.