Separation of a truncated protein by ion exchange chromatography

RNA Master Purple.Haze1st at gmail.com
Tue Aug 29 23:53:06 EST 2006


Alex писал(а):

> My protein expresses as two species with predicted pI=9.3 and pI=8.8
> due to truncation of 14 N-terminal residues (out of 400). I've tried to
> separate them by Mono-S column in a buffer with pH=7.5, but failed.
>
> QUESTION: Is it possible to separate such proteins by the ion exchange
> chromatography? Any suggestion about the buffer, pH, or other
> techniques excluding preparative isoelectrofocusing?

If the truncation variant is something you dont want then may be you
should try Mono-Q column instead. Or ammonium sulphate precipitation.
However, if you are 100% sure the truncation is your protein variant
and not some chaperone (a lot of chaperones from E. coli stick to
mono-Q) contaminant then it would be very very hard to separate those
two. You can try different expression hosts...

If the truncation variant is something you do want then just reclone
the truncation as DK suggested. However, you will still have the
problem with obtaining the bigger one as homogeneous fraction.

PH



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