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Purification of His tagged protein (from Pichia)

Randy Willis willis at gandalf.psf.sickkids.on.ca
Mon Apr 21 10:18:11 EST 1997


Ian,

While I do not myself work outside of coli with his-tagged proteins, I
am beginning to hear a lot of anecdotal material which indicates that
the contaminating proteins in eukaryotic systems are much greater than
in coli. Clearly, you're going to have to run a second, and maybe a
third, column to get clean protein.  I would only recommend gel
filtration as a step if the contaminants are clearly different in size
from your fusion protein.  Ion exchange or hydrophobic interaction are
probably your best bet and the pI of your protein and its stability in
high concentrations of ammonium sulphate will affect your selection of
which to use.  In fact, if it is stable, you may want to try an amm.
sulphate precipitation as a way to remove some of the contaminants. 
This is often used with the crude lysate and, I think, has been severely
underutilized in recent years.

Good luck,

Randall C Willis, Researcher
Biochemistry, Hosp for Sick Children
3522-555 University Ave
Toronto, ON
M5G 1X8  CANADA

416-813-5933 (ph)  -5022 (fax)

willis at gandalf.psf.sickkids.on.ca



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