Hi Angela,
I have had the same luck as you with HIC columns when I was isolating a
soluble protease a few years ago. I tried several different matrixes with
no improvement. I tried changing pH in 0.5 pH increments from 5.0 to 10.
No improvement. It seemed that no matter what I tried, my protease activity
would not elute from an HIC column. I could not ever determine if the
protein was sticking to the column irreversibly or if it was eluting in an
inactive form. Like you, I did ammonium sulfate precipitations prior to the
HIC column and I could see activity in the sample that I injected onto the
HIC columns, but I could never detect activity in any column fraction,
including flow through.
I wish I could give you some sort of salvation answer that would make this
work for you, but I can't.
The only advice I can give you is that there are hundreds, if not thousands,
of different matrixes to use in protein purification. When one does not
work, move on to another. Know the difference between being persistent in
making something work and wasting your time with something that will not
work. That is difficult.
If you know the enzyme, what else do you know about it? Use the known
properties of the enzyme to isolate it, i.e. does it bind metal (IMAC
columns), does it bind a substrate (substrate affinity column), are there
antibodies available etc.
Jim Lawrence
Pioneer Hi-Bred, Intl.
Johnston, IA 50131
515-270-5909
-----Original Message-----
From: Angela Merlo [mailto:angela.merlo@ioez.tu-freiberg.de]
Sent: Thursday, February 01, 2001 3:51 AM
To: Recipients of ABRF List
Subject: Hydrophobic Interaccion Chromatography]
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
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