Factor Xa Cleavage

[Paul Digard]

G E Martin wrote:
> 
> I planned to use Factor Xa protease to cleave one of my fusion proteins
> but initial results suggest that the protease has a preference for a
> non specific site in the fusion (cuts in hours) and very little for the
> engineered site (days). 

I recently made a fusion protein which cut well at the engineered Xa 
site, but also within the fusion moiety. I asked New England Biolabs 
about the specificity of Factor Xa, who replied that:

"Xa will cleave subsets of the Xa site such as EGR or GR.
This type of activity usually happens if the protein is denatured or
partially denatured. If the protein is badly denatured the Xa may 
cleave after Arginines alone."  

I'm thinking of swapping to a thrombin or enterokinase cleavage site 
if the uncleaved fusion checks out for activity.

On the non-cutting of the engineered site, my original protocol for 
pMAL expressions (from Biolabs I think, but the top page has 
gone....) says that it may be necessary to denature the protein with 
GuHCl to free up the cleavage site. I guess that's not likely to 
help the non-specific cutting, quite apart from whatever it does for 
any activity you might want from your protein.

Hope this is helpfull.


Paul Digard

Division of Virology
Department of Pathology
University of Cambridge