Dan Crimmins <crimmins@pathbox.wustl.edu>@aecom.yu.edu> on 02/01/2001
08:24:01 AM
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Subject: Re: Hydrophobic Interaccion Chromatography]
Amgela: One question, did the protein band of interest elute from the
column?? It is always possible that your protein is stuck on the column
and you may be able to knock it off with 20% ethylene glycol in buffer.
Alternatively, you can use water. I have seen this with several proteins,
esp ones that are greasy, I suspect that a bezoate dehydrogenase is fairly
large and probably a dimer and has very hydrophobic domains. One other
column you may want to try is hydroxylapatite (runs slow, but works like
magic sometimes) Use 10 mM Pi to load, and wash with step gradient of 10,
200 , 500 and 700 mM Pi at around neutral pH - I have use BioRAD
Hydroxylapatite for the purifcation of several dehydrogenases).
If you like the HIC, try loading in lower concetration of ammonium sulfate
and eluting with a gradient from 0 to 30 % ethylene glycol, after washing
the column with plain buffer. regards, gautam
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
>
>
>
>
Angela,
Does this enzyme have a multi-subunit structure? Perhaps the work-up
and
chromatography on HIC is causing dissociation of the subunits, eventhough
this mode of chromatography is considered "mild". Any cofactor(s) that may
have been lost during the purification? Try dialysis after the HIC step vs.
a buffer that your enzyme "likes" and add back any metals, etc. that may be
required for activity.
Regards,
Dan L. Crimmins, Ph.D.
Washington University School of Medicine
Department of Immunology and Pathology
Division of Laboratory Medicine
660 S. Euclid Ave., Box 8118
St. Louis, MO 63110
Phone 314-454-8514
Fax 314-454-5208
e-mail crimmins@pathbox.wustl.edu
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