Fusion protein cleavage

Rich Dudley rdudley+ at pitt.edu
Wed Dec 16 08:14:21 EST 1998


kll at crc.dk wrote:

> I am going to make a fusion protein for purification purposes and I would like
> to know about good and bad experiences with different types of cleavage
> enzymes. As far as I know the most popular proteases for cleavage are:
>
> Thrombin
> Factor Xa
> Enterokinase
>
> Can anybody recommend any of these (or other) enzymes with regards to
> effectivity, specificity, price, removal of protease etc.
>
> Thanks
> Kresten
>
> --
> Kresten Lindorff Larsen
> Dept. Yeast Genetics
> Carlsberg Laboratory
> Denmark
>
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Those three are pretty much it for specific cleavage of proteins.  Which one you
use will depend greatly on the composition of your protein and fusion partner.
Factor Xa is expensive in large quantities from any supplier.  Enterokinase (or
enteropeptidase) is relatively common but may contain some contaminants.  If
you're studying a pancreatic proenzyme (e.g., trypsin), you would want to avoid
enterokinase, since that also activates trypsin.  Thrombin is cheap, but has a
degenerate cleavage specificity.  You should examine your protein's AA sequence to
ensure there isn't a thrombin site in there.  There are a couple of tools on the
WWW for doing this, but if your fusion partner isn't too long, it's just as good
to do it by eye.  You can find a couple of references in Medline about thrombin's
specificity.

Good luck!

rich


--
--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdudley+ at pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
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