How did you check that the expressed protein is indeed your protein of interest?

Given that the Trx tag is at the N terminus than it is unlikely that translation will start downstream of it at your insertion site, it is more likely that what you see is the Trx tag fused to a part of your sequence. did you sequence your clone to make sure you have the full Trx-POI fusion all in frame?

The expressed protein has been checked by western blotting using the specific antibodies and found the band of the desired molecular weight.

Is it possible that Trx Tag may not be fused with the recombinant protein (POI).

Possible, but with an N-terminal tag not very likely unless you have a self cleaving site between the tag and your POI. Is it possible you accidentally cut out the tag when cloning your sequence into the plasmid? Another thing to check is whether you happen to have a good ribosom binding site between the tag and your sequence that can be used to initiate translation downstream of the tag.

Thank for your support, we are also thinking in the same directions.

But in an another case, a different gene was also cloned and expressed using this pET32b+ vector using different primers for gene amplification and different restriction enzymes, then again we found that the expression of POI with desired molecular weight and lateron confirmed by western blotting. In this case also, there is no addtional 109aa Trx Tag™ thioredoxin protein can be seen in the expressed protein.

So, is there any other possibility regarding problemtatic fusion of 109aa Trx Tag™ thioredoxin protein.