From: John Philo <jphilo@mailway.com>
  To  : Richard M Thomas <rthomas@ifp.mat.ethz.ch>
  Date: Wed, 27 Jan 1999 11:25:56 -0800

RE: His Tags

Richard,

I can't comment about specific issues for His-tagged proteins in the
centrifuge, but I will comment on some general problems with His tags
based
on experiences at Amgen and by some of our A.P.L. clients.

1) The His tag definitely can alter the conformation and bioactivity of
the
protein.  I know of cases where the stability of the native structure was
significantly altered, where the His tag clearly was binding back to the
protein, and where the His tag totally killed the bioactivity.

Even using a vector where the tag can be proteolytically cleaved does not
necessarily get you a protein with native activity, since this typically
still gives a non-native amino terminus (although this would probably
only
be true in cases where the amino terminus is particularly important for
activity).

2) In many cases His tags don't actually work well as purification tags,
and
generally require that the metal column step be done under denaturing
conditions.  One of our clients had been relying totally on His tags for
their primary purification and was unsuccessful with quite a few
different
proteins. (Even when they could purify them they were often inactive). 
With
our help they have now obtained >10 of these by conventional purification
methods and all are active.

3) On the other hand, it is not uncommon that the His tag increases
expression in E. coli significantly (total expression, not just the
soluble
fraction), so it can be useful in that regard even if you choose to
ignore
it as a purification tag.

John Philo
Alliance Protein Laboratories

-----Original Message-----
From: Richard M Thomas [rthomas@ifp.mat.ethz.ch]">mailto:rthomas@ifp.mat.ethz.ch]
Sent: Wednesday, January 27, 1999 8:55 AM
To: RASMB@bbri.harvard.edu
Subject: His Tags


Hello all
There have been mutterings, from time to time, on how protein
purification
methods involving *his* tagging can really bugger up centrifuge
experiments(among other things). With a view to creating a sort of
archive
of such stories, if any of you have horrors of this type to relate, I'd
be
glad to hear about them, whether published or otherwise.
All the best
Richard

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