His-tag protein purification

Bud Brown neur003 at uabdpo.dpo.uab.edu
Mon Mar 3 18:18:40 EST 1997


(I'm posting this for a friend who would appreciate advice from the net
community.  Thanks!  Bud Brown)
   I cloned a His-Tag fusion protein (which contains 4 disulfide
bridges)
and expressed it in AD494(DE3)PlysS e. coli using a Novagen expression
system.  This strain of e. coli (which has a thioredoxin B mutation)
should
allow the protein to form disulfide bridges as it is being expressed in
the
cytoplasm, and should thus render native protein.  There is some
evidence
that the expression system works as advertized and the expressed
protein
is native, but that after the protein is purified on a nickel column it
is mostly denatured.  The nickel column appears to have reduced the
disulfide bonds.

Have others encountered this problem before?  Could the nickel itself,
or
perhaps impurities in the column like copper or oxygen, have cause the
disulfides to isomerize on the column?  If so how do I get around this?



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