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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.

-Doc Sheldon-

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.
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