Enterokinase cleavage problem - HisTag (Jul/23/2005 )

Dear All,

Does anyone of you have ever faced the problem that the enterokinase cleavage site in the HisTag construstrion is not accessible for cleavage by the enterokinase?

We tried two different enzymes but without success. Can you think of any control HisTag construction we could aquire in order to confirm that our enzyme is working?

If it's not the enzyme, is there any other solution to overcome our problem?

Thanks for any input!

Freiberger

Novagen provides a cleavage control protein that is a high MW with an EK cleavage site in the center. You can run this on a gel before and after treatment to test your enzyme.
Try adding more enzyme than is suggested and letting the reaction go overnight. If your cleavage site is somehow obstructed, you may want to add a spacer of a few residues between the tag and the enterokinase site. Good luck!