FActor Xa Cleavage

pathos pathos at freerealtime.com
Tue Feb 29 13:12:27 EST 2000


In article <Pine.SOL.4.05.10002291114520.20363-100000 at granby>, Andy
Scotter <pcxajs at unix.ccc.nottingham.ac.uk> wrote:

>         Has anyone had any luck with Factor Xa cleavage kits?  I'm looking
> into cleaving a fusion protein with Factor Xa but I've heard that they
> aren't always successful (is anything?).  What size fusion proteins have
> been cleaved successfully?

The factor Xa cleavage site is rather poorly defined other than it cleaves
C-terminal to an arginine.  I can give you no less than 8 different
sequences that can be cleaved by fXa.  And all of them differ from the
thrombin cut site.  The size of your protein is not important.  What is
relevant is the 3d conformation of the cleavage site.  If it is buried
then factor Xa will not cleave well at all.  If I were you, I would find
another protease to use.  But if you insist on using factor Xa, try adding
PEG-7000(10% final concentration).  Remember that fXa is autoproteolytic. 
Once you have resuspended it, it must be used within about 10 hours or it
will chew itself up.

regards,

-- 
Peter Pediaditakis
University of Pittsburgh
Dept. of Pathology




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