[Q] Best Protease for Tag Removal

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Nov 17 16:36:25 EST 2000


Michael Oda <mnoda at lbl.gov> wrote:
:Hi,
:
:We are working with a His-tagged recombinant protein and would like to 
:cleave the tag off in a specific and cheap manner. We have been using 
:FactorXa and have found that the cost of cutting 10+ mg of protein to be 
:pretty high. I'm sure there must be a better enzyme out there for our 
:needs. We have tried Endopeptidase but it cleaves our target protein at 
:a secondary site.
:
:Someone suggested an enzyme called "iZyme". It was an unconfirmed 
:reference and I have been unable to identify a source for this enzyme. I 
:am hoping there is anyone that has heard of this apparently 
:recombinant/cheap/specific enzyme. If so, could you please send me 
:information on the supplier and its recognition sequence?

The best (most specific) protease for cleavage is TEV viral protease
from Gibco. I am pretty sure it will also be very costly to keep 
purchasing it for preparative scale cleavage. It is recombinant, so 
perhaps you can find  someone who can provide you with an expression 
plasmid or you can let some undergrad student to PCR it out subclone it. 

Of course it means you will need to redo your expression vector(s)
to include protease recognition/cleavage site.

:We have also been considering placing the amino acids Asp-Pro with their 
:acid labile peptide bond between the His tag and the target protein. 
:Does anybody have any experience with this particular peptide bond and 
:its acid lability? Fortunately our protein is rather durable and folds 
:back to a native conformation after 3M Guanidine treatment and appears 
:to be somewhat acid stable.

No idea how efficient it is. Perhaps worth trying but I'd be sceptical of
this approach. Acid+heat will, albeit with lower efficiency, hydrolyse 
other peptide bonds; you might end up with a mess that will require 
further purification; also, you might already have Asp-Pro in your 
sequence. 

        - Dima







More information about the Methods mailing list