Separation of a truncated protein by ion exchange chromatography

[Dr Engelbert Buxbaum]

Alex wrote:

> 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 you try a buffer of say, 8.8 or 9.0, the pI 8.8 protein will be
mostly uncharged or slightly negatively charged. It will interact only
weakly with MonoS. However, the pI 9.3 Protein will be more positive and
interact more strongly with the sulphonic acid groups of MonoS. 

Note that the statement "protein X is at a given pH 90% ionised" is
equivalent to the statement "each protein molecule is ionised 90% of the
time". owever the difference in pI and hence binding to the column
isnothat great and you will need a very shallow gradient to achieve to
separation. 

It may be more promising to use chromatofocussing, that is elution of
proteins with a descending pH-gradient. The pIs of the proteins should
be somewhere in the middle of the pH-range used, so a pH 11-8 gradient
should work in your case. Note that the gel matrix needs to have an even
buffering capcity over the entire pH-range, the same is true for the
eluting buffer. Pharmacia (now part of GE unless I blinked and missed
another change of ownership) is one company offering suitable systems
(Polybuffer).