klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu (Dima Klenchin) wrote:
>skumar at mbu.iisc.ernet.in ("S. Kumar Singh") wrote:
>:Hi all,
>:Is there any good established procedure for removing His tag from the N
>:terminus of a protein., I would greatly appreciate a reference on this.
>:Thanks a million in advance.
>>Only if you have protease recognition site between the tag and
>the protein. Most conventional proteases are not specific enough
>and cut expressed protein as well, however. TEV protease and
>its 6 aa recognition site appears to be the best choice. Look up
>Gibco catalog for more details.
Deep down in my mind there is something about inteins (self-splicing
proteins) with a his-tag, fused to the protein of interest: Bind it to
a Ni-column, induce splicing activity and elute clean, tag-free
protein; then clean your column by eluting the remaining intein with
imidazole. Unfortunately, I don't remember a manufacturer name or
reference.
Bye, Frank
