There could be more than one reason fo this observation.

After the cleavage of the GST tag, the protein's sequence could lead to an anomalous migration in SDS-PAGE (+/- 5 to 10 kDa difference in the expected vs. observed MW is not rare).

Or the cleavage after the GST may not be only site that is cleaved, there could be another site cleaved by the added protease (or proteases from the lysate). This should be something that is time-dependent, so after shorter incubation you should observe more than one band (full-length and shorter version). In any case, you should confirm the size and identity of that band.

Another reason could be something that is associated with the GST-protein e.g. a chaperone like DnaK (which happens to be around 40kDa in size), and while you cleave your protein, it could become insoluble without GST and only the soluble chaperone stays in solution and with the GST-protein removed, it could remain the only protein in your "purified" fraction.

Hope that helps.

Hüseyin Besir

Thank you for your answer. Can you help me with a reference for the following part explanation?

Another reason could be something that is associated with the GST-protein e.g. a chaperone like DnaK (which happens to be around 40kDa in size), and while you cleave your protein, it could become insoluble without GST and only the soluble chaperone stays in solution and with the GST-protein removed, it could remain the only protein in your "purified" fraction.

Hi, I'm sorry for the error in my post. I wanted to write DnaJ, not DnaK. These two chaperones are similar to the Hsp70 (DnaK) and Hsp40 (DnaJ) system in eukaryotic cells. This could be checked by MS analysis if avaliable.