stabilizing enzyme during storage

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Sun Jun 30 16:32:07 EST 2002


You need to shed some light on what is it that causes loss of activity. You
need some biophysical data on kinetic profile deterioration upon storage,
dependance on temperature & pH, aggregation profile, proteolysis check
(protease inhibotors - do they help at all?), effect of metal ions, etc.
Without this, you're groping in the dark. How pure is your protein to start
with - single step Ni-NTA purification can result in as low as 90% purity,
which means that you can have proteases galore as well as all sorts of other
nasty things in your sample...
Also, what do you call 'fully functional' - do you have a reference to
natural-source enzyme ? If you do, then how was activity preserved in that
case ? If you do not, how do you know that you are purifying 100% active
enzyme ?

Good luck,

A.G.E.
"Pedro Rocha" <p.s.c.rocha at durham.ac.uk> wrote in message
news:3D1F6F6C.3100E69B at durham.ac.uk...
> Hello,
>
> I would appreciate any input from protein experts on how to stabilize a
> purified
> protein (obtained by overexpression in E.coli).
>
> This particular enzyme is purified using Ni-NTA resin, being eluted with
> 20 mM
> imidazole, 300 mM NaCl and 50 mM NaH2PO4. The enzyme is fully functional
>
> immediately after purification. However, it is very unstable. Storing,
> even for a
> few hours at 0-10 C  (ice or fridges) knocks-out most of the activity.
> Same for
> -20C. Addition of various %s of glycerol did not help at all. I would
> greatly
> appreciate any suggestions of additives that could be tried to stabilize
> the
> enzyme.
>
> Any suggestions for additives that may also stabilize it during
> functional
> assays are welcome. I realize that this is potentially a more tricky
> challenge
> (the colorimetric assay is limited in terms of pH and salts etc.).
> Stabilizing it
> during storage would be a great step.
>
> Thank you,
>
> Pedro Rocha
>
>





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