From: Richard M Thomas <rthomas@ifp.mat.ethz.ch>
  To  : RASMB@bbri.harvard.edu
  Date: Fri, 05 Feb 1999 13:12:10 +0100

HIS tags - a summary

Thanks to all who have contributed to my HIS-tag appeal. I will try to
summarize what has been sent in, and hope that I get it right.
	In general, negative experiences seem to outweigh positive, especially in
ultracentrifuge experiments. This is particularly true in cases where the
protein is known (or suspected) to exhibit association/aggregation
phenomena. Purification considerations may favour the incorporation of the
tags, but here, too, there has been negative feedback.
	The rationale for the effects reported is not absolutely clear and may
vary from case to case.  The induction of incorrect folding, due to the
introduction of non-native sequence is probably part of the answer, and
might well lead to alterations in activity, aggregation etc. Specific
association, mediated by the tag itself and leading to changes in
aggregation state, is almost certainly also implicated, and this may be
sensitive to experimental conditions. The tag, as an independent entity,
may interact with the rest of the protein, producing structural change.
There is also evidence for post-translational modification in the HIS tag
itself.
	In brief, HIS-tagged proteins should be treated with caution. While
changes in a protein's structure obviously have implications for the
analysis of data obtained on it by any experimental technique, it may well
be that it is the poor, long-suffering hydrodynamicist who is first
confronted with unravelling the protein's real physical behaviour in
solution. You have been warned.

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*	(Tom Laue) Negative experiences with MyoD, leucine zippers, DSPG1,DSPG2,
proprietary proteins. Aggregation problems with EPCR were relieved after
removal of the tag. Aggregation was not metal ion-dependent, pH sensitivity
unknown. Green fluorescent protein and two proprietaries behaved well.
*	(Jack Correia) Kinesin constructs exhibited aggregation, although, as the
non-tagged versions were not investigated, the origin of the effect remains
unclear.
*	(John Philo) General points: (i)HIS tags alter conformation and activity.
The tag may bind back on the protein, killing activity. Cleaving the tag
does not necessarily restore activity  (ii) HIS tags do not always work
well as purification accesssories and the conditions required may lead to
deactivation (iii) However HIS tags may increase expression and might be
useful for this property alone.
*	(Walter Stafford) The HIS-tagged monomeric extracellular domain of
c-erbB-2 behaves well, even at high protein concentration
*	(Frank Whitby) The crystal structure and monomer/dimer equilibrium of
uroporphyrinogen decarboxylase is unaffed by HIS-tagging.
*	(Knut Langsetmo) Reports aggregation in HIS-tagged proteins from
experiments that do not involve centrifugal techniques.
*	(Kieran Geoghegan) Unexpected extra mass in fusion proteins in E.coli is
due to modification of HIS tags. Apart from anything else, this may lead to
purification difficulties on ion exchange chromatography.
*	(Richard Thomas) HIS-tagged Lysine hydroxylase is (apparently) tetrameric
in the presence of DTT but dimeric in its absence - odd in itself. Cysteine
probably not involved. Non-centrifugal experiments indicate a pH
sensitivity of association in the HIS-tagged form, and current theory
suggests that the behaviour may be due to metal ion-mediated association,
via the tag. A comparative study of tagged and untagged protein is underway.

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References received:-


Kieran F. Geoghegan et al. (1999) Spontaneous a-N-6-Phosphogluconoylation
of a "His-Tag" in Escherichia coli. The Cause of Extra Mass of 258 Da or
178 Da in Fusion Proteins.Analytical Biochemistry, February 1st issue

Whitby et al (1998) EMBO May 1st issue


Rosenfeld et al  (1996)  "Structural Studies of Kinesin:Nucleotide
Intermediates", J. Biol. Chem., 271, 30212-30221.

Correia et al (1995)   "Sedimentation Studies on The Kinesin Head Domain
Constructs K401, K366 and K341.  Biochemistry, 34, 4898-4907.

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Hope this is of some use. All the best to everyone

Richard

Dr Richard M Thomas
Inst. für Polymere
ETH-Zentrum
CH-8092 Zürich
Switzerland
(tel) +41 1 632 5540
(fax) +41 1 632 1073