Protein Purification Problem

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Thu Dec 14 10:44:36 EST 2000


Jonathan_Kurtis at Brown.edu (Jonathan Kurtis, MD/PhD) wrote:
:Hi all,
:
:I have purified a recombinant fusion protein which contains 1167 aa with a
:calculated Mw of 118 kDa. I intend to cut this fusion protein with Factor
:Xa protease into the fusion partner (619 aa, 53 kDa, pI=5.95) and the
:protein of interest (548 aa, 65 kDa, pI= 6.18). 
:
:These fragments differ in size by only 20% and in pI by 0.23 units. There
:are no affinity tags on the fragments.

Then what is the fusion partner if not affinity tag? Why not run 
cleaved protein mixture over the same column you purified
original protein and collect your fragment of interest in flow 
through? 

:Using a starting material of 500ug/ml, should it be possible to separate
:them by ion exchange (merits of anion vs cation??) ?? 

Ion exchange will work if you find proper conditions. Look up 
titration curves and pick a pH where the two posess most
differing charges. Play with salt in loading and elution. Anion 
vs cation depends on your proteins and their charges. Strong
ion exchange (Q, S) vs weak ion exchange (DEAE, CM) 
depends on your protein "stickiness" and stability (some big 
proteins get "crucified" on strong ion exchangers).

:by size-exclusion
:chromatography??

Very unlikely.

:I need to purify approximately 25 mg.

Then I'd pack a column of ~ 20 ml (assuming dynamic
separation conditions).

Also, chromatofocusing can be used. It will resolve 0.2
pH unit easily and cleanly as long as your protein does
not precipitate at pI and/or in ampholytes solution.

        - Dima






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