Do proteins self-digest?
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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