We have a MalE-target protein fusion with an enterokinase cleavage site between the two domains. The most effective cleavage was achieved at 0.4 ug protease per mg of fusion protein using a 16 hour incubation at 37C. The cost of the enterokinase is prohibitively expensive for production of 1 g of protein. Short of building a new expression vector, has any one had success in modifying the reaction conditions of enterokinase to increase the proteolytic efficiency? If so, what did you do? Thanks.