Cleaving Fusion Proteins - (Sep/04/2009 )

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 anyone had success in modifying the reaction conditions of enterokinase to increase the proteolytic efficiency? If so, what did you do? Thanks.

The Invitrogen enterokinase manual suggests increasing the concentration of CaCl2 and/or Tween-20 in your reaction buffer (maximum of 10mM CaCl2 and 1% Tween-20 in your 1x buffer). I've not used yet so take with a large grain of salt.