Hi Adam,
I have not read all the thread as it came all at once and late (9:00pm
here).
I believe the best way to strip a protein of metals is to first adsorb
it onto a solid support (e.g. IEX) and then use a sufficiently low-pH
(say equal or below 6) buffer that contains also EDTA.
You will probably need several washes but it works!
Also be aware that EDTA binds well to several proteins.
HTH,
Nadir Mrabet
Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
N-gere - INSERM U-954
University of Lorraine, Nancy
School of Sciences and Technologies
& School of Medicine
9, Avenue de la Foret de Haye, BP 184
54505 Vandoeuvre-les-Nancy Cedex
France
Phone: +33 (0)3.83.68.32.73
Fax: +33 (0)3.83.68.32.79
E-mail: Nadir.Mrabet <at> univ-lorraine.fr
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On 19/05/2014 20:21, Adam Brummett wrote:
Thank you everyone for the comments and suggestions. To answer a few
questions:
-I do not use a treated buffer system. I have just used the nano-pure
water. I have looked into Chelex, but before I bought it I wanted to
see if you all recommended it. I was trying to avoid this, but it may
not be possible now.
-the active site does bind metals and is promiscuous in binding, so it
is not know if the His tag or active is the source of contamination,
but cleavage is not an option for us. The biding of metal is going to
be needed for phasing, so good point Tim, hopefully just not in the
His site.
-thank you Vivoli for the protocol, seems very thorough. Have you had
success with it? I anticipate I'll need to go down this road.
-Roger, the metals you mentioned (Zn and Fe) are the problem and I
expect to have to go to heroic measures to get an apo enzyme . But you
did mention easier ways of getting metal substituted. I have some
evidence that I can do this. Do you have any other thoughts on this
matter? Maybe a reference to something similar (non-apo but could
substitute?
Thank you all so much for the help and advice.
-Adam
On May 19, 2014, at 12:55 PM, Roger Rowlett <rrowl...@colgate.edu
<mailto:rrowl...@colgate.edu>> wrote:
The answer depends on a number of questions:
* What metal ion are you trying to eliminate?
* What kind of metal-binding site is involved?
o A peripheral or loose binding site? (e.g. surface calcium
ions)--these may respond to chelators
o An active site coordinated metal? (e.g.,
metalloenzyme)--these can be refractory
Many metalloenzymes are not going to give up their metal to
chelators, or just any chelator, or at all. Denaturation, dialysis,
and refolding is an extreme way of removing metal ions to make
apoprotein. Won't work for every protein. Chelation can be highly
specific, that is one chelator may work, while another, similar one,
will not.
Some metal ions are notoriously difficult to eliminate, because they
are adventitious trace contaminants in nearly everything, e.g. zinc
and maybe even iron. (Plastic-ware seems to be often loaded with
trace iron, and also is capable of adsorbing metal ions form
solution.) To make apo-enzymes from zinc proteins, you have to go to
heroic efforts to ensure that glassware, water, buffers, and reagents
are zinc-free, especially if you don't have high (mM) concentrations
of protein to work with.
A His-tag is very likely to snag adventitious metals from solution,
and can often mess up metal analysis for metalloproteins by providing
"extra" metal. If this is a problem for your application, you may
want to consider removing the His-tag.
If you are making apoenzyme to get a different metal installed
(metallosubstitution), there are slightly easier ways to do that than
going through the apoenzyme route.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote:
Hello All,
I apologize for the non-crystal related question. I am trying to get a fully
metal-free apo enzyme. The 6x His construct is consistently purified with some
metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA
and DFO and then dialyzing it away, but this has shown little to no effect. Any
thoughts or recommendations would be greatly appreciated. Thanks.
Adam
