His tagged protein expression

Mark T. Worthington mtw3p at AVERY.MED.VIRGINIA.EDU
Thu Aug 6 15:59:31 EST 1998


The polyhistidine tag can cause oligomerization of proteins which may
not be completely eliminated by boiling in SDS-loading buffer and will
show up on gels as a higher order product.  Often this is a ladder. 
Since these tags will coordinate a variety of transition metals, such as
Cu, Co, Zn, etc., it can occur from your bacterial lysate and does not
have to be blamed on leaching from the column.  Since the tag is
ostensibly an alpha helix, it can bind several metal atoms in free space
using the IMAC matrix and whatever metal ions it brought with it.  We
had a protein that we had purified for NMR using this method, and
routinely had to drop the pH to 1-2 to get the metal off before
HPLC--the trifluoroacetic acid in the solvents wasn't enough.  Also, if
you cleave off your His-tag or dialyse against EDTA, the effect should
go away, as ours did.  That assumes your protein doesn't require a metal
cofactor for activity.

This property was described a while back (Euro J Biochem 1991 by Lilius
et al) using Zn in a soluble chelate to EGTA,  and recently rediscovered
in a different form in Biotechniques 25:20-22 (July 1998).  

Best of luck; hope this helps.


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