Phoebe,

It just so happens that we've been working on a C4 Zn-finger (or something
very much like a Zn-finger) very recently without knowing that we had one
(it's in the PheRS from S. haemolyticus, link to paper below) and we've
purified the protein w/o any issues on an IMAC column (His-SELECT). When we
found the metal site in the structure, we sent the protein out for metal
analysis (since we did not know for sure what metal(s) occupy the site) and
the Zn was all there within 10% error margin (based on expected molar ratio
of 1:1, protein quantity by necessity estimated from protein concentration),
so no exchange with Ni or other metals took place.

This follows the trend I noticed with other C3 and C4 Zn-fingers - they
generally seem to bind Zn so tightly that no appreciable exchange takes
place under 'normal' conditions. C2H2 may be different - I have no direct
experience with that sub-class.

Again, only based on my personal experience (literature cases probably exist
that indicate otherwise), Zn(CX)4 chelate does not seem to bind to Ni-NTA,
His-Select, or Talon resin. Incomplete assemblies (i.e. Zn-free) may bind
which is why it's probably a good idea to add a tiny amount of Zn into the
lysis buffer, to make sure that all the incompletely chelated species are
'filled in'. Again, based on personal experience - these 'empty' species
tend to form nasty aggregates which fall out of solution ASAP, anyway.

Please note that the above is applicable to Zn fingers that supply all four
ligands from the same protein chain. When two chains supply the ligands,
many more nasty things become possible.

Hope this helps,

Artem

P.S. the paper is linked to http://www.xtals.org/pubs.html, first link in
the Anti-bacterial section.

-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
[EMAIL PROTECTED]
Sent: Thursday, January 03, 2008 7:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Zn fingers and Ni columns

This has probably been discussed before, so apologies in advance.
We're eyeing a protein that has a probable C4 Zn finger in the 
middle.  The collaborators who are nicely going to PCR it up want to 
know if we'd like it with or without a His tag.
Is it a bad idea to co-mingle Zn-binders and Ni columns?  Or is it 
likely to bind the column quite nicely without the tag?
         thanks,
         Phoebe


----------------------------------------------------------------------------
-----------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alpha
betically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html 

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