native HisTag elution alternative

Fred dynamin at hotmail.com
Tue Oct 31 22:39:04 EST 2000


I just read that L-histidine can work well.  In theory it sounds like
what you need.  The reference is at a a rather annoying web site
(BioMedNet, but maybe it will work for you):
http://research.bmn.com/tto/categories/record?uid=TTO.elstto00_01689525_42_t01389&node=TOC%40%40TTO%40001%40103%40001_103&tocname=Purification+methods&tocnode=TOC%40%40TTO%40category%40purification_methods

Or briefly..
Purification of a hexahistidine-tagged protein using L-histidine as the
eluent

Steve Gort a s-maloy at uiuc.edu and Stanley Maloy b
[a] Steve Gort[b]Stanley Maloy, Department of Microbiology, University
of Illinois, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin
Avenue, Urbana, IL 61801, USA.

The His·Tag protein purification system enjoys a great deal of
popularity due to its ease of use in rapidly preparing large amounts of
purified protein. The His·Tag system takes advantage of the affinity of
histidine residues for metal cations, usually divalent cations such as
Zn2+, Cu2+, or Ni2+ (Ref. 1).

A stationary support, in this case chelating Sepharose, is charged with
the metal salt of choice. A crude cell extract is then applied to the
column and only proteins with a high affinity for the divalent cation
will bind to the column. After washing to remove other proteins, the
bound protein is eluted off the column. Most commercial systems use
imidazole as the eluent to remove bound protein from a column.
Alternative approaches for elution have been reported, including
lowering the pH or the addition of EDTA (Ref. 1, 2). Anecdotal evidence
suggests that many His·Tag proteins that retain activity in vivo are
inactive in vitro after elution. The loss of activity with imidazole as
the eluent could be due to an effect on the protein. The use of low pH
might interfere with protein activity by denaturation and EDTA could be
binding important metal co-factors. Here, we describe a less harsh
option for the eluant, L-histidine.

Phil

(Don't reply to this email.  I am not "Fred".  Thats what the newsgroupd
are for...)



soenke behrends wrote:
> 
> Dear netters,
> 
> we purify a protein containing a HisTAG with
> a Ni-NTA matrix and elute the protein with
> imidazole (250mM). Unfortunately enzyme activity
> of the eluted protein is strongly inhibited by
> 250mM or 150mM imidazole, whereas 50mM imidazole
> lead to 50% of the activity without imidazole.
> 
> We now want to turn to an alternative elution
> approach e.g. EDTA. Has someone made good ex-
> perience with this under native conditions and
> could provide us with a protocol?
> 
> I have also been thinking about whether one could
> use high concentrations of DTT. In the manual
> (Quiaexpressionist) they write that DTT can reduce
> Nickel thereby preventing nickel from binding His-tagged
> proteins. High concentrations of a reducing agents
> would be good for the protein anyway, but it is not
> mentioned as an elution strategy.
> 
> I am not so keen on low pH for elution, since I fear
> that it will not be any good for the protein. But I
> have so far no evidence on that.
> 
> We would really appreciate any comment or tip
> Thanks for your time and help
> Soenke






More information about the Methods mailing list