RE: [ccp4bb]: Protein crystals with a 10 His tag?

["Bernhard Rupp" <br@llnl.gov>]

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> -----Original Message-----
> Tim Grüne
> Sent: Friday, November 30, 2001 12:01 PM
> To: Narayanan Manoj
> Subject: Re: [ccp4bb]: Protein crystals with a 10 His tag?

> know, His-tags are believed to be unstructured and flexible, i.e., they
> inhibit crystallisation.

I think it is a bit more differentiated. If everything that is unstructured
and flexible would inhibit crystallization, we would not have any structures
in the pdb that don't show defined N (40%+ or so) or C termini (20%?).

One could argue that if it *prevents necessary crystal contacts from being
formed*
it is bad. Unfortunately, we lack predictive power wrt crystal contacts,
even given a
structure model.

A tag also could prevent proper folding if the termini are buried. This
appears statistically to be more likely for C termini than N. Given a
model, you could look at that. Novel structure, up the creek.

Anekdotal case examples exist either way, we had a 68 residue tag on a
(large)
protease and it diffracted to 1.8A, others report even the vicinity of his
tag
plasmid containing vial in the same room as crystals jinxes theirs.

All of the above translates into we have no clue, because we seldom hear
about
negatives, which we would need to establish proper statistics. So maybe
after
unbiased Structural genomics data bases have been filled, we may be able to
give a
likelihood of reduced/increase success of his vs non-intagged constructs.

A (probably outdated) collection wrt His tag successes can be found

http://www-structure.llnl.gov/internal/his/histag.htm

The ease of purification is perhaps worth it, also there are some systems
(R-TEV) that cleave the histag off and leave only one residue of the
TEV site on the nterminal but I am no expert.

BR

Therefore, the shorter the better, but this must
> not always be true. The charged tag may as well positively alter your
> proteins behaviour.
> Tim
-----------------------------------------------------------------
Bernhard Rupp
Macromolecular Crystallography and Structural Genomics
LLNL-BBRP L448                               Phone (925) 423-3273
University of California                     Phax  (925) 424-3130
Livermore, CA 94551                          email    br@llnl.gov
URL                                 http://www-structure.llnl.gov
TB Structural Genomics Consortium  http://www.doe-mbi.ucla.edu/TB
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