I expressed a protein in SUMO vector that express SUMO fusion protein, so, I wanna do an enzymatic essay after the protein purification without cleavage the SUMO protein. Has someone experience in enzymatic essay without cleavage the SUMO fusion protein?
Thank you!
Vania Sedano
Does your protein form dimers to be active? It could be that the SUMO tag prevents dimerization. Do you know where the protein's active site is located? If the active site is sterically close to the N-terminus and you have an N-terminal SUMO tag you could run into problems. Try moving the tag from N to C or vice versa.
Hello Cornelia,
Yes, the protein form an homodimer. SUMO tag is in N-terminus and the active site is in 260 aminoacid in primary structure.
It is important to look at the folded protein, the sequence gives you only limited information. The SUMO tag can interfere with dimerization and/or with the active site. Is there a crystal structure available for your protein?
Hi Vania,
it sounds, how should I say, highly unlikely that the active site is 260 aa. I agree with Cornelia, you should reason on the crystal structure, even better if your protein was crystallized with SUMO tag. Are you running your enzyme assay with a control protein with no tag? Are you confident that your protein is otherwise properly folded? Do you have additional information on dimerization (e.g. SE-UPLC), secondary (CD) or tertiary (DSC) structure of the protein?
Without homologous crystal structure, it is unpredictable if the SUMO tag wll interfere with your enzymatic assay even if the active site is 260aa away from the N-terminus while your SUMO tag is at the N-term, because after protein folding the active site might still be too close for substrate binding,etc. I strongly suggest you remove the SUMO tag with proper protease before conducting your assays.
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