Hello everyone,
I expressed a fusion protein using pet32a and purifing it by IMAC. But when this protein was cleaved by EK enzyme at temperature 23℃ for 16hr,the objected protein(about 15KD)was degradation to some little molecular weight .So i reduced the enzyme cleavage time to 2hr, the protein degradation still exists. I also try to change the cleavage temperature to 4℃,but it does not work. Could anyone please give me some idea and suggestions how to solve the protein deagradation after EK cleavage?

Did you check if you have the site for EK clevage inside your protein ? (asp-asp-asp-asp-lys) If is yes, bad luck you are going to get the same degradation because your protein have the site. If not, check the conditions and try to do as similar as possible to the recomende conditions by the manofacturer
Good luck