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 -
Dave Schooley's suggestion notwithstanding, I'm not going to advise you to
passivate your frits because I doubt you're using Sepharose in a
high-pressure column.
Your observations - good behavior in salting out but difficult elution from a
HIC column - are characteristic of a protein that's water-soluble but has
major hydrophobic patches on the surface of its tertiary structure. We had a
similar problem with Interleukin-2. In order to get it to elute from a HIC
column, we had to use our least hydrophobic HIC material (PolyMETHYL
Aspartamide) and to include 5 or 10% propanol in the final mobile phase.
Interleukin-2 eluted at the end of the gradient, albeit in high yield. I
suggest you determine if your enzyme will retain its activity in the presence
of 5% propanol, then try some version of our procedure.
Best regards,
Andy Alpert
PolyLC Inc.
9151 Rumsey Road, ste. 180
Columbia, MD 21045
tel: (410) 992-5400 FAX: (410) 730-8340
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