cleavage of MBP fusion proteins

[Mark Brandt, Ph.D.]

In article <32222346.7A7F at slip.net>, GBasi at slip.net wrote:

> We have repeatedly attempted to cleave the maltose binding protein from 
> our fusion proteins of interest using factor XA and found that the 
> cleaved MBP no longer binds to amylose.  Has anyone out there 
> encountered this same problem and if so, how were you able to overcome 
> it?  (P.S.  The fusion protein itself is able to bind amylose resin 
> before cleavage, just not after cleavage with factor XA).
> please reply to gbasi at slip.net,  thanks for your help.

I am not sure that I can really answer this -- Factor Xa doesn't cleave my
construct effectively. Using other cleavage methods (trypsin or
hydroxylamine) I have gotten more complete cleavage. Following cleavage
the MBP tends to bind the amylose resin poorly, but this may be due to the
presence of maltose in the binding site; if I subject the MBP (or fusion
protein) to more than one chromatography step in between amylose columns,
I usually get good binding of the MBP to the amylose, so I suspect that
the MBP is somewhat reluctant to release the maltose.

To answer your "overcome it" question, I generally have better results
separating the MBP from my (cleaved) protein by gel filtration, rather
than amylose chromatography.

-- 
Mark Brandt, Ph.D.
My opinions are my own, but I tend to give them away to anyone who fails to flee fast enough.