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
>>
