This can be very hard to do because quite a few proteases are promiscuous and will cut substrates solely based on masking of the polypeptide within the structure of the protein. Typically these proteases will not stop cutting at a single nick - they often proceed until they can't 'dig into' a buried or obstructed section. Often one protease nicks the chain and other proteases (or amino/carboxy peptidases) extend the gap. I would base the search on the observed phenomenon - i.e. is it a single nick, a few residues missing, or a whole swath or domain?
Theoretical aspects (i.e. searching for sequences) have been mentioned already - practical ones of course include fractionating the environmental factors (i.e. cell juice) where the cutting takes place and exposing the uncut substrate protein (if you can get it!) to fractions, then sub-fractionating; you can use class-specific inhibitors to further narrow down the selection of enzymes; in some cases a trap (crosslinking, suicide substrate, etc.) can be used to identify the perpetrator. In some cases it may be possible to identify the enzyme by separating all possibilities on e.g. a 2D gel then exposing a fluorogenic peptide to the gel and trying to find a glowing spot (or spots). But again, this is not a very clean solution due to promiscuous nature of many proteases and the sad fact that post-gel renaturation is not guarranteed (not to mention that the protease in question may be multisubunit, or activated by cofactors and gel will destroy these interactions). What do you actually see? Artem On Thu, Mar 31, 2011 at 8:59 AM, Brett, Thomas <tbr...@dom.wustl.edu> wrote: > Hi all: > I was wondering if anyone had any tips on identifying proteases. I have a > protein for which I know the proteolytic cleavage site. What are the best > ways to identify the protease that does the cutting either: > 1) bioinformatically (i.e., a good database to search using the cleavage > site or a consensus) > 2) experimentally (some engineered substrate to trap/identify the substrate > or any other method?) > Thanks in advance > -Tom > > Tom J. Brett, PhD > Assistant Professor of Medicine > Division of Pulmonary and Critical Care > Washington University School of Medicine > Campus Box 8052, 660 S. Euclid > Saint Louis, MO 63110 > http://brettlab.dom.wustl.edu/
