In article <nef-0102951725000001 at moose.bioc.cam.ac.uk> nef at mole.bio.cam.ac.uk (Nick Fisher) writes:
>From: nef at mole.bio.cam.ac.uk (Nick Fisher)
>Subject: Re: Do proteins self-digest?
>Date: Wed, 01 Feb 1995 17:25:00 +0000
>In article <Pine.3.89.9501301624.A28223-0100000 at ccshst01.cs.uoguelph.ca>,
>aiyo at uoguelph.ca (Abiye Iyo) wrote:
>> Hi,
>> I recently used the GST fusion system to purify my protein (a
>> cellulase). Everything went fine up to the stage of gel filteration and
>> I had a nice single band . Now the misery part is that my protein which
>> is about 57kD degrades to two low molecular weight products which are
>> still active on zymograms. Does this mean my prep is contaminated with
>> proteases or I am experiencing some form of self digestion judging from
>> the fact that my protein was pure initially? And how can I prevent
>> this? I need help before my patience runs out.
>>>> Abiye
Yes, for example calpain, when exposed to calcium, first activates by
autoproteolysis and then, if exposure to calcium is prolonged, digests itself
into inactivity. The way to prevent this is with a calcium chelator such as
EGTA. You also must consider bacterial contamination and endogenous proteases.
Gary
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Gary Krause (gkrause at cms.cc.wayne.edu)
Department of Emergency Medicine
Wayne State University, Detroit, MI USA
