N-acetyl transferase purification, What to do next?

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Tue Mar 9 10:39:16 EST 1999


In article <36E02370.324E5276 at hotmail.com>, Biology <mpiraee at hotmail.com> 
wrote:
>Hi evryone
>It is a long time I am trying to purify N-acetyl transferase from
>Streptomyces venezuelae. It works on a specific substrate p-nitro phenyl
>serinol. I fractionated the crude cell extract by 60% amonium sulfate
>(after removing the pellet fro 30% am. sulfate) which is highly active.
>Then on DEAE collumn it was again fractionated by NaCl gradient. I found
>the active fraction with the highest activity. I analyzed that fraction
>with HPLC. It showed still it has two more proteins with very close MW.
>I already tried HIC columns but it did not worked. I need the peptide to
>be sequenced
>Any commnet on how to proceed for further purification is welcomed.
>

It is a standard practice to do higher resolution ion exchange
after initial crude purification. If you have an access to FPLC
or HPLC, do chromatography on Mono Q - it's a different 
chemistry and has very high resolution. It is likely to help.

Alternatively, try hydroxyl apatite chromatography. (I'd
recommend ceramic HA from Bio-Rad). There is no reason
high resolution HIC should not work too. Just play with
conditions... Finally, high resolution chromatofocusing 
(FPLC/MonoP) virtually gurantees you a nice separation
if you are lucky and your ptotein does not precipitate
during it.

Hope this helps,

        Dima



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