Q. regarding stabilizing enzyme (for storage)

D.K. dk at no.email.thankstospam.net
Sun Jun 30 20:45:43 EST 2002


Pedro Rocha <p.s.c.rocha at durham.ac.uk> wrote:
>Hello,
>
>I would appreciate any input from protein experts on how to stabilize a
>purified
>protein (obtained after 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.

If you can come up with an explanation as to what it is about it that 
makes it unstable, the decision might be a lot more rational. Generally  
speaking you have plenty of things to try: immediate neutralization 
to pH > 7.0, protease inhibitors, sugars (sucrose, trehalose, xylitol), 
detegents (triton X-100, chaps, octyl-glucoside), betaine, DTT ...
the list is endless. If it can work for you, perhaps easiest solution
is to "drop-freeze" in LN2 (25 ul drops directly into LN2; you end
up with plenty of beaded protein aliquotes that you can store at -80C).

DK




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