HI, I want to crystallize a 18 kDa protein with His-tag & HA tag on C-terminal, and a 19 amino acid signal peptide on N-terminal. My protein is a Singel-Domain antibody. I don’t know if they effect on its 3D-structure. My protein with condition has activity. I can’t decide that I can crystallize it or I must separate its tags and signal peptide. Please help me.
Hi Sara Canon
- Test Crystallization with Full-Length Protein: Sometimes, crystallization can still be achieved with tags and signal peptides, especially if the protein folds properly and retains activity. If you’re not sure, you can try crystallizing the full-length protein first.
- Remove the Tags and Signal Peptide: If crystallization fails with the full-length protein, consider removing the His-tag, HA-tag, and signal peptide. Removing the tags might improve the quality of crystals, as the purified protein would only consist of the functional part, potentially improving its overall structure and stability.
- Use a Protease Cleavage Strategy: If removing the tags and signal peptide is critical but you still need them for purification, you can use a protease (such as TEV or thrombin) to cleave them off after purification. This way, you can purify your protein with tags and then remove them before crystallization.
Since your protein has activity under the conditions you’re working with, it seems promising that the overall structure is intact, but whether tags will affect crystallization can vary. You may need to experiment with both the full-length and cleaved forms.