6 replies to this topic

[aj_xy999]

Hello Forum,
i'm looking for some protocols / kits /  information on efficient removal of 6xHis tag fused to a recombinant protein at the C-terminus.
cheers!
Ajay

[InvisibleSurfer]

I think you are gonna ave to go back and use a different vector to clone your gene into, that has a cleavage site (usually thrombin) between the his and the mcs.

[Mbiologist 1]

HI,
Invitrogen has a kit called EKMax or any Enterokinase (Novagen) will cleave the His-tag from your protein. then try Enterokinase Resin to remove the enzyme from the protein reactions. under all cases, you will need to run a pilot experiment to optimize the amount of Enterokinase you need to cleave your fusion protein.

Good Luck.

[wirly]

Enterokinase has a recognition site (Asp-Asp-Asp-Lys) at which it cuts preferentially.  Unless you have this site next to your His-tag you are still out of luck like the surfer said.

Edited by wirly, 01 September 2004 - 01:12 PM.

[pesji]

Hi all, new to the forum I am Pesji working in the STI in Basel

Quiagen has an enzyme to effitiently remove his tag at least they clamed it does so. It's called Agilent his Tag removal and it use an enzyme called  TAG zyme DAPase unfortunately it's quite expensive and I was looking for cheaper solution

If anyone can help

Anyway here is the LINK


Pesji

[Luke]

aj_xy999, on Aug 16 2004, 01:07 AM, said:

Hello Forum,
i'm looking for some protocols / kits /  information on efficient removal of 6xHis tag fused to a recombinant protein at the C-terminus.
cheers!
Ajay

Hi, we have the same problem and I must tell you that TAG Zyme by qiagen is useless too since it cuts only at the N-ter (indeed it's tagged at the c-ter).
I heard that even if you put a cleavage site between your protein and the 6XHis at the C- ter, will be nearly impossible to cut out the tag because all the protease have quite big problem to cut out a c-ter end of a protein... Anybody can confirm this?
Thank's
Luke

[Ali]

Hi...
You can fuse your DNA target into pGEX-4T-2 vector and trombin can be use to remove GST from your recombinant protein after purification in GST column.
Good Luck