Factor Xa cleavage of a pET42 Fusion protein

[Michael Sullivan]

I've made a GST fusion protein in pET42. The resulting protein should  
consist of GST-His Tag-thrombin cleavage site-S Tag-Factor Xa  
cleavage site-My target protein.  I'm trying to do cleavage of the  
fusion with Factor Xa. That seems to be working, except I usually get  
a doublet around the size of the expected protein. The lower band of  
the doublet seems to comigrate with a "native" non-tagged version of  
my target protein as expressed from a different pET construct. In  
cleavage reactions with increasing amounts of Factor Xa, it appears  
that the larger band of the doublet is an intermediate to the shorter  
version (i.e. with more Factor Xa, I see less of the larger band and  
more of the smaller band) I haven't yet carried out cleavage under  
conditions where I see only the lower band cleavage product, however.  
My question is whether anybody has seen this sort of behavior before.  
I realize that Factor Xa and other "specific" proteases can cleave at  
secondary sites but I don't really see any good candidates N-terminal  
to the "real" Factor Xa site. I wonder if maybe the Factor Xa might  
be cutting after the arg in the pET42 encoded thrombin site, since it  
might be fairly exposed due to the His tag immediately N-terminal to  
it. I'd feel better if I knew what both bands were. Any ideas?

Thanks,

Mike Sullivan
---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)