Dear all, I would like to share with you some problems I am experiencing with a protein, in case someone has an idea about what it is going on:
I am working with a protein module that gets trapped in Ni-NTA or glutathione beads. If the protein is purified without tag (ion exchange plus gel filtration), the protein can be purified properly. However, when expressing with a His or GST tag, it gets trapped and it cannot be eluted with imidazole/glutathione or even with 8M Urea (in case it is unfolded).... Does someone know what can be happening? We do not have any information about an enzimatic activity of the module that might permit the protein bind covalently to the beads.... Furthermore, we got the crystal structure in complex with another protein and the folding of the module does not resemble anything with enzimatic activity.... Many thanks in advance for all your suggestions, Maria
