Do proteins self-digest?

larson eric elarson at ux1.cso.uiuc.edu
Mon Jan 30 18:21:16 EST 1995


aiyo at uoguelph.ca (Abiye Iyo) writes:

>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.
========
 
Sounds like protease contamination.  Try adding inhibitors to the mol. wt.
col. buffer -- they can be dialyzed away later.  Mol. wt. separation is
usually time-intensive, affording ample opportunity for residual proteases
to function.  Couple this with the absence of other proteins (likely if
this is the second step with the first being ion-exchange) and you have
perfect conditions for protease problems.  Proteolysis on mol. wt. resins
has affected me directly.
 
-- 
Eric Larson                  | University of Illinois at Urbana-Champaign
USDA/Agronomy                | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu     | Voice 217.244.3079  Fax 217.244.4419
Fidonet: 1:233/4.1           | My opinions are my own, but correct :-) 



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