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They have: Primary site: The designed cleavage site Secondary sites: Weaker recognition sequences elsewhere in your protein Non-specific activity: Especially at high concentrations or long incubations Thrombin is the worst offender: Research on thrombin-induced degradation found that "while thrombin and factor Xa are quite specific for cleavage at the inserted cleavage site, proteolysis can frequently occur at other site(s) in the protein of interest." The problem is compounded by commercial thrombin preparations containing secondary protease activity from contaminating proteases

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