Angela;
I do not have a concrete answer for you, just some wordy suggestions because I
like enzymes. Enzymes are wonderful things with individual personalities. As
is with people, no two are alike. I always envy someone who is enjoying the
beginning of an enzyme purification.
Anything may happen to your protein in enzyme purification, especially if your
protein interacts with a natural partner through hydrophobic interactions. On
the other hand, it may change it protein-protein interactions or aggregation
state during purification, both of which can alter its apparent charge,
apparent isoelectric point, solubility, and stability. So the performance in
anyone step is no assurance of future success in other steps.
What is useful upfront is a pilot study of enzyme solubility for the crude
enzyme at different pH's, a few buffer systems, and ionic strengths. This is
easily done with dialysis in the right buffer to mimic the duration of a
purification step where enzyme inactivation may occur, do centrifugation, then
assay. Your assay is easy, so the study is expedient. For example, if your
enzyme activity in gone after overnight in a low ionic strength buffer at pH 8
but not at pH 6, it is useful information. Do this with as much protein
solution as you can afford so that there is enough material to form aggregation
as may occur when the enzyme is more concentrated. I use 20 mL per condition
at times. Otherwise, I repeat the solubility study when the enzyme is highly
concentrated such as after an ammonium sulfate concentration step.
Next I would put the enzyme through a simple binding and elution test for a
number of chromatography resins by a batch procedure using simple spin columns
in small volumes. Assay right after elution, and after 24 hours. This defines
what is allowable in the crude state prior to enzyme environment modification
by the purification steps. It may recommend the order of your purification
steps. With your good assay, this should take about a day. The speed also let
you test whether a step is usable if you work really fast, and enable you to
test how to stabilize the enzyme right at elution.
Since you can do an ion-exchange step successfully already, next I would like
to know the apparent size of the enzyme in size exclusion chromatography in a
couple of buffer conditions, after the ion-exchange step. This is to test how
easily the enzyme may form aggregates. Do this on a SMART system if possible
so it only takes two hours each.
Now you are in a position to trouble shoot the phenyl-Sepharose step or to
avoid it all together. This step removes over 90% of the protein
contaminations in a hurry and is a drastic environmental change for your
enzyme. I have seen enzymes that precipitate in that step, or aggregate badly,
or inactivate because of irreversible conformation change induced by the
hydrophobic interaction mimicking its natural interaction with a protein
partner. The hypothesis being that some enzyme interaction with its
protein/DNA partner may happen as a one way reaction when strong hydrophobic
forces are involved. That happened to two of my enzymes. There are other
exotic inactivation mechanisms that can occur in this step due to activation of
proteases that are previously kept inactive by their natural inhibitors that
are now stripped off by the hydrophobic interaction resin. It did happen to
one of my enzymes. Hydrophobic interaction is a powerful step for some
enzymes, but not for many enzymes.
Do you really need the ammonium sulfate for your enzyme to bind to
phenyl-Sepharose. If not, your binding may be too tight for easy elution. You
may have to let your gradient end as plain water or 2 mM Tris, or a long
isocratic wash with 10 mM Tris at the end of the gradient. You may wish to
test a less hydrophobic resin like octyl-Sepharose.
Lastly, the pH optimum of an enzyme is not a reliable guide to the pH for
ion-exchange chromatography. You will learn something about the apparent
isoelectric point in above solubility test. I would do ion-exchange with pH on
both sides of the isoelectric point, or at two pH units apart, and you will see
interesting swapping of protein elution profiles you can customize to your
enzyme and the key contaminants.
Hope this is helpful.
Tony
At 09:50 AM 2/1/2001 +0000, Angela Merlo wrote:
>
>Hi everyone,
>I try to purify an enzyme (bezoate dihydrodiol dehydrogenase) from
>Rhodococcus opacus (Gram +).
>I noticed that the optimal pH for this enzyme is 9, so I started an
>anion exchange chromatography with buffer pH 8 because it was supposed
>that 9 is too high. The chomatogram was nice and i got activity in a
>fraction. The problem came afterwards when I tryed a Hydrofobic
>Interaccion Chrom. I precipitated the protein pool with (NH4)2SO4 and
>resuspended them with puffer pH8+salt. The activity was high so I did a
>HIC (phenyl sepharose) with decreasing concentration of TrisHCl 25mM pH8
>+ (NH4)2SO4 1M. I got a good chromatogram but no activity in any
>fraccion. Then I tryed a butyl Q-sepharose column and also didnĄt work.
>Last time I used a buffer pH 7,5 and I had again no activity.
>Do you have any idea where the problem can be?
>
>Thanks
>
>Angela
>
************************************
Anthony T. Yeung, Ph.D.
Director, Fannie E. Rippel Biotechnology Facility
Member, Institute for Cancer Research
Fox Chase Cancer Center
7701 Burholme Ave., Philadelphia, PA 19111
Voice: 215-728-2488 FAX: 215-728-3647 email: AT_Yeung@FCCC.edu
Public Key: D994 4658 6AE2 12A6 A538 D18C 461F 5ED2 21C5 2AFF
http://www.fccc.edu/research/labs/yeung/
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