His-tag: C-terminal cleavable through protease? - (Jul/30/2007 )
I'm constructing a protein with a his6-tag. Therefore I wonder if its possible in principle fuse the histidines onto the C-terminus and in between a proteolytic cleavage site for such as Thrombin i.e. Very widely used are pET28 vectors but there is no version with a c-terminal cleavable his6-tag. Is there a good reason NOT to have a proteolytic cleavage site in the C-terminal region of the protein of interest?
Did anybody already construct this fusionprotein: |N-terminus|-protein + cleavage site + his6-tag-|C-terminus| ?
I used His-tag constructs many times and I never saw the one you ask for. I guess the explanation would be that if you only have 3 his left to bind your protein of interest to Ni....your life gets more complicated! I had problems with one of the protein complexes that I was trying to purify because the HIs-tag was mostly buried inside the structure, so it depends on your protein. Have you though about using another tag like TAP?